Did Itachi know that Shisui had a Mangekyo Sharingan? Don't take me for a fool, I know the situation, but I've been watching the fight between Itachi and Sasuke, where he said something like this from the old times: "Come after me, when you will obtain the same eyes as mine, being the third to hold a MS."

He already had the Mangekyo Sharingan, so we can safely assume that he knew the story about his "elders", hence knowing not just about Madara, but Izuna and of course Shisui too. Obviously, he didn't know about Obito/Kakashi, but is this a plot hole, or did Itachi really not know that along with Madara and himself, two others already possessed that kind of Sharingan, making his statement false, since Sasuke would've been at least the fifth person?

(I've written Shisui for purpose in the title, but we don't just have to talk about him.)
It is not a plot hole. And he isn't necessarily wrong. He just meant the third person alive. Of course he knew about Shisui, he had his eye. Of course he knew about Izuna and Madara too since he introduced their story to Sasuke. And he probably knew about other unnamed Uchihas too, if there were any, because as he said the Uchihas were killing their best friends for decades to achieve it. So I can't find any other logical explanation.