The aim of this study was to investigate the role of E6/E7 mRNA in discriminating patients who were high-risk human papilloma virus-positive associated with cytology-negative and Atypical Squamous Cells of Undetermined Significance (ASCUS). This study comprised of 380 women (age: >30 years) who were associated with high risk of cervical virus infection and they underwent simultaneous examinations of cytology, Human Papilloma Virus (HPV)-DNA, E6/E7 mRNA, and colposcopic pathological biopsy. The end-point of the study was set as the histological confirmation of high-grade Cervical Intraepithelial Neoplasia (CIN) II or higher (II+). In group HPV16/18-positive patients Negative for Intraepithelial Lesion or Malignancy (NILM), after E6/E7 mRNA discrimination, the positive predictive value (PPV) of CINII+ was increased from 21.62% to 40.54%, and the difference was statistically significant (χ2=4.40, P<0.05). Meanwhile, the Negative Predictive Value (NPV) of E6/E7 mRNA was as high as 97.30%. In other high-risk (HR)-HPV-positive patients with ASCUS, after E6/E7 mRNA discrimination, the PPV of CINII+ was increased from 16.18% to 23.81%, and although the difference was obvious, it was not statistically significant (χ2=0.98, P>0.05). Meanwhile, the NPV of E6/E7 mRNA detection was as high as 96.15%. E6/E7 mRNA can better discriminate the HPV16/18-positive patients with NILM from other HR-HPV-positive patients with ASCUS.