The ark was built by Christians who said they believe the biblical story was a historical event.
Critics have said the attraction should not have received tax incentives as it contradicts science education.
The ark is based on a biblical story of a man who received an apocalyptic warning from God about a massive flood.
Answers in Genesis, the ministry that built the ark, said the ship's dimensions are based on those described in the Bible.
The ark is also 85ft (26m) wide and 51ft (15m) high, according to the group.
The ark, which is about 40 miles (64km) south of Cincinnati, Ohio, in Williamstown, features displays of animals in cages as well as exhibits on the group's theories on the world's existence.
The group believes that God created everything, including dinosaurs, about 6,000 years ago.
Scientists have said the dinosaurs became extinct about 65m years before man appeared.
Ken Ham, president of Answers in Genesis, said the total cost of the ark is more than $100m (Â£77m).
The attraction has been at the centre of controversy in Kentucky, where it was awarded a tax incentive in 2014 worth up to $18m (about Â£14m).
The incentive allowed the park to recoup taxes on money made from visitors.
The tax break was then revoked after it was revealed that Answers in Genesis would only hire Christian staff members. However, the group sued and won.
Opponents of the theme park told the AP it is a violation of separation of church and state.
"Basically, this boat is a church raising scientifically illiterate children and lying to them about science," said Jim Helton, who lives about a half-hour from the ark.
One of the park's biggest critics is science advocate and former television personality Bill Nye "the Science Guy".
Nye debated Mr Ham on evolution in 2014, attracting millions of viewers online.
The video of the debate posted on YouTube by Answers in Genesis has more than five million views.
Following the debate, Mr Ham announced that a bond offering from Williamstown had raised $62m (about Â£48m) for the project, allowing the group to break ground on the site a few months later.
Answers in Genesis estimates that the theme park, which debuted to the public on Thursday, will draw about two million visitors in its first year.

Omar Gonzalez, 42, made it past the front door and all the way to the executive mansion's East Room before being tackled.
As part of a plea deal, he will serve between 12-18 months in prison and be barred from entering Washington DC.
He was previously ordered to undergo a 30-day mental health evaluation.
Gonzalez will be sentenced in June.
The breach was led to the resignation of the director of the Secret Service, the agency responsible for protecting the president.
"We are pleased that Mr Gonzalez has chosen to take responsibility for his incomprehensible decision to leap a fence and charge into the White House with a knife," US Attorney Ronald Machen said in a statement.
"He is lucky to be alive," Mr Machen said, adding the US anticipates he will be required to undergo psychiatric treatment.
Gonzalez scaled the White House fence on 19 September, dashed across the lawn and gained entry through an unlocked door, then barrelled past a guard and ran into the East Room before being tackled.
Eight hundreds rounds of ammunition, two hatchets and a machete were found in his car, officials say.
Gonzalez, an Iraq War veteran said to suffer from post-traumatic stress disorder, was previously stopped by Virginia police in July.
Officers found two powerful rifles, four handguns and other firearms and ammunition in his vehicle along with a map marking the White House.
In addition, it has been reported Secret Service agents interviewed Gonzalez twice during the summer but concluded he was not a security threat.

Determined to not repeat their first innings collapse, Lightning batted with resolve on a placid Contarf pitch.
North Down leg-spinner Jacob Mulder (2-62) struck twice in the morning session to offer the Knights some hope.
But Lightning skipper John Anderson and Simmi Singh negotiated the final two sessions with a century partnership.
Despite following on from a first innings deficit of 294 runs, Leinster were never in trouble as Anderson and Singh produced the best partnership of the match with an undefeated 146.
Only two wickets fell all day as the home team accumulated 221 in 91 overs.
A theme park in the US state of Kentucky has unveiled a 510-foot-long (155 metre-long) wooden model of Noah's Ark.
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An Iraq war veteran who jumped the White House fence and ran into the building with a knife has pleaded guilty to two charges.
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Leinster Lightning produced an improved batting performance in their second innings against Ulster Knights to keep their unbeaten Inter-pro record.