HeLa　cells　are　a　commonly　used　cell　line　in　many　biological　research　areas.　They　are　not　picky　for　culture　medium　and　proliferate　rapidly.　HeLa　cells　are　a　notorious　source　of　cell　cross-contamination　and　have　been　found　to　be　able　to　contaminate　a　wide　range　of　cell　lines　in　cell　culture.　In　this　study,　we　reported　a　simple　and　efficient　method　for　detecting　the　presence　of　HeLa　cell　contamination　in　cell　culture.　HPV-18　was　used　as　a　biomarker.　The　cell　culture　supernatant　was　used　directly　as　the　template　for　nested　PCR　without　extracting　nucleic　acid.　By　PCR　amplification　of　the　cell　culture　supernatant　with　the　designed　primers,　we　were　able　to　detect　the　presence　of　HeLa　cells　in　the　culture.　The　sensitivity　of　this　method　can　reach　1%,　which　is　10-fold　higher　than　Short　tandem　repeat　sequence　(STR)　profiling.　This　simple,　rapid,　and　＂noninvasive＂　quality　checking　method　should　find　applications　in　routine　cell　culture　practice.