Aim To prepare and identify the monoclonal antibodies against recombinant protein of chlamydial protease-like activity factor from chlamydia pneumoniae for clinical diagnosing C.pneumoniae infection. Methods The chlamydial protease-like activity factor recombinant protein was used as antigen for the immunization of female BALB/c mice, and the spleen cells of mice were fused with SP2/0 myelomas. Indirect ELISA and Western blot analysis were used to screen the hybridomas secreting antibodies and then subcloning positive clones were carried out to establish stable cell lines by limiting dilution. Ascites were induced to produce MAbs and their specificity were identified by indirected immunofluorescence (IIF) test and Western blot analysis based C.pneumoniae type strain. The new indirect ELISA was estabilished to examine coronary artery plaques from patients and the results were analyzed by statistical method. Results Four hybridoma cell lines secreted the monoclonal antibodies stably were finally obtained and named as 3F8、7B9、8C4 and 11B5, respectively the subtype of the monoclonal antibodies secreted by 3F8 and 7B9 strain was IgG2b, the other two monoclonal antibodies were IgG1 and their titers in ascites were 1∶28000, 1∶16000, 1∶38000 and 1∶12000, respectively. The Western blot and IIF analysis showed that these monoclonal antibodies have excellent specificity to C.pneumoniae. The results of detecting clinical samples by the new indirect ELISA based on self-produced the monoclonal antibodies had better consistency with the results of PCR kits for C.pneumoniae infection. Conclusion The specific monoclonal antibodies against chlamydial protease-like activity factor recombinant protein of C.pneumoniae were obtained. All the 4 monoclonal antibodies belonged to IgG, which can react with native chlamydial protease-like activity factor from C.pneumoniae. The self-producted monoclonal antibodies are suitable for C.pneumoniae antigen diagnosis to coronary arteriosclerosis patients.