Equine influenza (EI), caused by the equine influenza virus (EIV), is an acute respiratory disease that has become enzootic worldwide, resulting in frequent outbreaks and substantial economic losses within the equine industry. In this study, we developed a competitive enzyme-linked immunosorbent assay (NP-cELISA) for the detection of antibodies against the EIV nucleoprotein (NP). The assay was designed by coating plates with purified monoclonal antibodies (mAbs) against the NP protein, followed by simultaneous incubation of the test serum samples and HRP-NP antigen in a competitive binding reaction. Receiver operating characteristic (ROC) curve analysis demonstrated that the assay achieved 100% sensitivity and specificity. To assess the diagnostic performance of the NP-cELISA, we evaluated 119 clinical samples in parallel using the NP-cELISA, a commercially available competitive ELISA (ID.vet-cELISA), and the hemagglutination inhibition (HI) assay as the reference standard. The results indicated that the NP-cELISA showed an 87.4% concordance rate with the HI assay, outperforming the 78.2% concordance rate observed between the ID.vet-cELISA and the HI test. Additionally, in a serological surveillance study conducted using the developed NP-cELISA in China from 2021 to 2023, equine serum samples showed an average annual seroprevalence of 37.96% for EIV antibodies. In conclusion, the NP-cELISA developed in this study demonstrates significant potential as a reliable and efficient diagnostic tool for the serological detection of EI, with broad applicability in various settings.