AbstractQuality control is very important during the development of 3‐valent (16/18/58), 9‐valent (6/11/16/18/31/33/45/52/58), and 15‐valent human papillomavirus (HPV) vaccines (6/11/16/18/31/33/35/39/45/52/56/58/59/68). All 3‐valent, 9‐valent, and 15‐valent HPV vaccines contain the HPV16 antigen; therefore, a detection method that can specifically identify HPV16 in vaccines is urgently required. This study aimed to develop and characterize monoclonal antibodies to assemble a highly specific HPV16 detection kit. The HPV16 L1 pentameric protein developed as an immunogen was used to prepare monoclonal antibodies. From the pool of prepared monoclonal antibodies, we selected 4G12 and 5A6 to screen and evaluate their subtypes, specificity, neutralizing activity, serum competition, binding affinity, and gene sequencing. After these characterizations, an enzyme‐linked immunosorbent assay kit for these monoclonal antibodies was developed, and excellent quality was demonstrated in the assessment of linearity, repeatability, and specificity. The developed detection kit has great potential for wide use in clinical testing and quality control in vaccine production processes.