Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.