Coxsackievirus A16 (CV-A16), one of the primary etiological agents of hand, foot, and mouth disease (HFMD), causes outbreaks of the disease in young children worldwide. Researchers conducted this study to promote the prevention and control of HFMD. The research and development of the CV-A16 vaccine have been carried out in China. However, lacking a recognized CV-A16 antigen detection method, the evaluation and QC of vaccine effectiveness are minimal. In this study, we established a quantitative enzyme-linked immunosorbent assay (Q-ELISA) to determine the antigen concentration in CV-A16 vaccines applied in manufacturing in China. A neutralizing antibody 16E1 was used as a capture antibody to bind to various CV-A16 antigens of different subgenotypes. An antiserum from CV-A16-immunized rabbit conjugated by HRP was suitable for detecting and quantifying CV-A16 antigens. The Q-ELISA was validated for specificity, linearity, accuracy, precision, and robustness using the CV-A16 antigen NS. Furthermore, we utilized the Q-ELISA to quantify antigen contents of vaccine bulks from six manufacturers and other intermediate products from one manufacturer. The results indicated that the Q-ELISA could satisfy the requirements of QC for all manufacturers involved.