Objective? To analyze the application value of rapid PCR detection in cell culture medium contaminated by bacteria and microorganisms. Methods? Materials for this study a human contaminant HeLa cell line（Hela cells）was selected，and the cell class microbial universal primers used 16 S ribosomal RNA（16S rRNA）gene sequences，PCR amplification of 16 S rRNA gene sequence fragments，set PCR mode to detect the contamination of cultured cells，and using this method，the contamination status of this cell line was examined in detail.Results? In the culture supernatant of HeLa cells artificially contaminated with Escherichia coli，Pseudomonas aeruginosa，Staphylococcus albicans，both amplified size fragments were present and corresponded to the fragment of interest.Of the 20 cell strain culture supernatants in this study，five amplified large and small fragments.Conclusion? This study established a universal primer PCR method for 16S rRNA gene sequence，which can quickly and effectively detect bacterial microbial contamination in cell culture，and has important application value for early detection of contamination.