Objective It has been well accepted that human papillomavirus type 16 (HPV16) is associated with human cervical cancer and HPV16 L1 protein could induce both humoral and cellular immune responses. The objective of this study is to map epitopes on HPV16 L1 protein and to provide information to the design of HPV16 prophylactic peptide vaccine.Methods The epitopes on L1 protein were screened by polyclonal and 2 monoclonal antibodies (B8 and F4G3) against HPV16 L1 protein from a 6-mer phage display epitope library with the method of immuno-affinity screening (Bio-panning). After 3 rounds of Bio-panning, the positive phages were detected by L1 antibodies again with ELISA. The positive phages reacted strongly with L1 antibodies were then identified by DNA sequencing.Results Three mimotopes have been screened by polyclonal and two monoclonal antibodies. The mimotope (LSLFSC) which reacted with monoclonal antibody B8 showed 50% homology with the sequence 270～275a.a (DSLFFY) of prototype HPV16 L1. Another mimotope (LTSSYS) which reacted with polyclonal antibodies had 66% homology with the L1 sequence 516～521a.a (TTSSTS), also a mimotope (DRWDRF) was found to have the homologic RF with the known L1 sequense 441～446a.a. The mimotopes LSLFSC and DRWDRF were adjacent to the epitopes at 267～269a.a and 422～441a.a reported by other researchers previously.Conclusions Our results suggested that there might be a batch of epitopes on HPV16 L1 protein, and the predominant epitopes of HPV16 L1 protein were located in the above two domains. These results would be heplful for the design of HPV16 prophylactic peptide vaccines and HPV polyvalent vaccines.