Abstract: Objective: This study was to establish a immuno in situ PCR method for the diagnosis of HCMV active infection. Methods: The cells infected with HCMV or renal tissues sections of paraffin embedded were used. A mouse monoclonal anti HCMV primary antibody and biotinylated secondary antibody are stepwise immobilized. After that, free streptavidin and a marker biotinylated pBV220 DNA are immunobilized consecutively. A segment of the immunobilized DNA are amplified by in situ PCR with the appropriate primers. Antigens induced by HCMV were visualised with a purple blue precipitate obtained. Results: The strong positive signals can be detected as early as 8 h postinfection by immuno in situ PCR, the positive signal was much more intensive with infection time. 6 of 23 case renal tissues of congenital malformed foetus were shown to be positive. Conclusion: This immuno in situ PCR assay was sensitive, specific and exact location and can be used in diagnostic laboratories to detect active infection caused by HCMV.