Abstract: The recombinant HPV16 L1 capsid protein was expressed in Escherichia coli, and the process parameters of cation exchange chromatographic purification of the recombinant protein were optimized by design of experiments（DOE） to obtain a higher recovery rate, which provided guidance for product production. First of all, the expression vector pET-30 a-16 L1 was constructed and transformed into E. coli BL21（DE3） cells. Recombinant HPV16 L1 protein was expressed in E.coli and fermented in a 50 L fermentor; Then four cation exchange chromatographic media were screened by single factor variable method. Design of Experiment（DOE） was used to optimize the loading flow rate and the system buffer pH. Purification of protein by hydrophobic chromatography was used in the second step to purify HPV16 L1. We analyzed the protein purity, recovery rate, endotoxin, host residual protein and residual DNA of recombinant HPV16 L1 virus-like particles（VLPs）. We observed HPV16 L1 VLPs with TEM; Finally, after the mice were immunized with HPV16 L1 VLPs, the level of neutralizing antibodies induced in the body was evaluated. The results showed that a buffer system with a pH of 8.0 and a sample flow rate of 5 mL/min were screened to perform POROS^TM XS cation chromatography as the protein purification conditions. The protein was further purified by hydrophobic chromatography; The purified protein concentration was 1.0 mg/mL, the recovery rate was 46.23%, the purity was 97%, the endotoxin content was less than 12.5 EU/mL, the host residual protein was 6.00 ng/mL, and the host residual DNA was 3.30 ng/mL. Six weeks after immunization in mice, the average of neutralizing antibody titer in serum Log 10 was 4.01. In this study, HPV16 VLPs were expressed by E. coli, and the purification process of cation exchange chromatography was optimized, which provided ideas for the development of virus-like particle vaccine and laid a foundation for the application of DOE in biological engineering.