Abstract: Objective To analyze the application value of nucleic acid detection of thinprep cytologic test(TCT)combined with nucleic acid detection of high-risk human papillomavirus(HR-HPV)in screening of cervical cancer. Methods The results of nucleic acid of HR-HPV and histopathology test in 332 cases confirmed as atypical squamous cells of undetermined significance(ASC-US)by TCT were retrospectively analyzed. These confirmed patients were divided into young group and middle and higher aged group according to age. The results of HR-HPV and cervical histopathology were compared between the two groups. Results The patients confirmed as ASC-US by TCT combined with positive HR-HPV had significantly higher histopathological coincidence rate of than those confirmed as ASC-US by TC combined with negative HR-HPV and those without ASC-US combined with positive HR-HPV(P<0.001). The positive rates of HR-HPV nucleic acid and histopathology tests in the young group - showed significant differences compared to those of the middle and higher aged group(P<0.05). There were no significant differences in the accuracy, sensitivity and positive predictive value of nucleic acid test of HR-HPV in ASC-US patients of two groups(P>0.05), but there were significant between-group differences in the specificity, Jordan index and negative predictive value of HR-HPV nucleic acid test(P<0.05). There was significant difference in the HR-HPV positive rate of different levels of cervical lesions in the whole group, the young group or the middle and higher-aged group(P<0.05). Conclusion The combination of HR-HPV improves the detection rate of cervical squamous intraepithelial lesions in ASC-US patients confirmed by TCT, and it is especially suitable to middle and higher aged group.