Enteroviruses (EVs) are the major etiological agents of hand, foot, and mouth disease (HFMD), which could lead to severe complications and even fatalities. Owing to the alternating or co-circulation of multiple EV serotypes and the lack of cross-protection, global institutions are pursuing the development of multivalent HFMD vaccines. Notably, the advancement of innovative multivalent HFMD vaccines into the clinical stage highlights the urgent need for a standardized platform to assess immunogenicity. In accordance with international regulatory guidelines, a standardized neutralizing antibody (NtAb) assay for CV-A10 is established through rigorous selection and systematic optimization of key assay components, representing the first such assay tailored for clinical immunogenicity evaluation of CV-A10-based vaccines. By integrating the 1st Chinese National Standard (NS) for CV-A10 NtAb and a standard detection virus, this assay exhibits enhanced detection sensitivity and significant reduction in the inter-laboratory geometric coefficient of variation (GCV), thereby providing a robust, reliable, and transferable platform. This assay facilitates accurate evaluation of multivalent HFMD vaccine efficacy, improves comparability across different vaccine products and provides genuine efficacy in real-world, serving as a valuable reference for global regulatory authorities.