question: Enterovirus D68 (EV-D68) is an emerging respiratory pathogen since the 2014 outbreak in USA. A low level of virus circulation has been reported in Kuala Lumpur, Malaysia in the past. However, the extent of the infection in Malaysia is not known. In the present study, we determine the seroepidemiology of EV-D68 in Kuala Lumpur, Malaysia before and after the USA outbreak in Aug 2014. A luciferase-based seroneutralization test was developed using a clone-derived prototype Fermon strain carrying a nanoluciferase marker. We screened the neutralization capacity of 450 serum samples from children and adults (1-89 years old) collected between 2013 to 2015. EV-D68 seropositivity increased with age, with children aged 1-3 showing significantly lower seroprevalence compared to adults. Multivariate analysis showed that older age groups 13-49 years (odds ratio [OR] 4.78 [95% CI 2.69-8.49], p<0.0001) and >50 years (OR 3.83 [95% CI 2.19-6.68], p<0.0001) were more likely to be EV-D68 seropositive than children <13 years. Sampling post-Sept 2014 compared to pre-Sept 2014 also predicted seropositivity (OR 1.66 [95% CI 1.04-2.65]). Presence of neutralizing antibodies against EV-D68 in the study population suggests that EV-D68 was circulating prior to 2014. A higher seropositivity post-Sept 2014 suggests that Malaysia also experienced an upsurge in EV-D68 infections after the USA outbreaks in Aug 2014. A low seropositivity rate observed in children, especially those aged 1-3 years old, suggests that they are at risk and should be prioritized for future vaccination. This article is protected by copyright. All rights reserved.