Developing a new method to isolate and proliferate Chlamydia pneumoniae is meaningful. This new method begins with isolating peripheral blood mononuclear cell from blood samples, and use polyethylene glycol to break peripheral blood mononuclear cells which are positive with Chlamydia pneumoniae antigen, then centrifugate these broken peripheral blood mononuclear cells together with Hep-2 cells. After 7-day′s culture, these cells were broken by freeze-thaw cycle, and put into new Hep-2 cells, then centrifugated together to finish the first passage of Chlamydia pneumoniae. The second to the fourth passages were conducted in the same way. At the same time, we used Micro-immunofluorescence and PCR methods to detect Chlamydia pneumoniae in Hep-2 cells, also used a genus-specific, fluorescein isothiocyanate-conjugated monoclonal antibody to Chlamydia lipopolysaccharide, to stain inclusions, and got the IFUs counts of both imported and isolated strains after each passage. With the method of Micro-immunofluorescence, we found that Hep-2 cells centrifugated with peripheral blood mononuclear cells, Hep-2 cells of the first passage, Hep-2 cells of the second passage were all strong positive with Chlamydia pneumoniae antigen, Hep-2 cells of the third passage was positive, and the fourth passage negative. We also detected Chlamydia pneumoniae DNA existing in Hep-2 cells of the second passage with the method of PCR. So this simplified way can successfully achieve the isolation of Chlamydia pneumoniae from peripheral blood mononuclear cells. The phenomenon of degeneration appeared in the fourth passage of isolated strain, which was still superior to imported strain, but it will not largely affect the culture and passage of Chlamydia pneumoniae from peripheral blood mononuclear cells.