Enterovirus D68 (EV-D68) is an emerging respiratory pathogen since the 2014 outbreak in the United States. 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 United States outbreak in August 2014. A luciferasebased 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 and 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% confidence interval [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-September 2014 compared to pre-Sept 2014 also predicted seropositivity (OR = 1.66; 95% CI = 1.04-2.65). The presence of neutralizing antibodies against EV-D68 in the study population suggests that EV-D68 was circulating before 2014. A higher seropositivity post-September 2014 suggests that Malaysia also experienced an upsurge in EV-D68 infections after the United States outbreaks in August 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.