Abstract: Objective To prepare John Cunningham virus (JVC)-like particles and establish a serological detection assay for JCV,and understand the seroepidemiology of JCV in general population in China. Methods The synthesized sequence of VP1 was inserted into the prokaryotic expression plasmid PET-21a, the resulting plasmid was transferred to the Escherichia coli BL21 (DE3), and then the protein expression was induced with IPTG, the expression products were analyzed with SDS-PAGE. Two steps of ultracentrifugation were utilized to purify the VP1 protein, the JCV-like particles were analyzed with transmission electron microscopy and hemagglutination assay. Then the purified JCV-like particles were used as antigen to establish the indirect enzyme-linked immunosorbent assay (ELISA) to detect the anti-JCV antibody in serum from general population. Results The results of transmission electron microscopy and hemagglutination assay demonstrated the shape and hemagglutination activity of JCV-like particles were similar to the natural JCV particles. ELISA was established by using JCV-like particles as antigen to screen the antibody to JCV. The seropositive rate of JCV in general population was 54.2%. Conclusion JCV-like particles were successfully prepared, the established ELISA could be used to analyze the seroepidemiology of JCV.