Objective To investigate the accordance of Hybrid Capture-II (HC-II) and real-time fluorescent PCR in detecting high-risk human papillomavirus (HR-HPV) DNA in cytological cervical samples and to investigate the sensitivity and negative predictive values of the two assays in screening high-grade cervical intraepithelial neoplasm. Methods HR-HPV DNA was in parallel detected with HC-II and real-time PCR in 877 cytological samples from women underwent liquid-based cytological test. For the patients with the abnormal cytological results or positive HR-HPV DNA, biopsy was performed. Results The two HR-HPV DNA detection methods showed concordant results in 804 of 877 cervical samples (91.7%, Cohen’s Kappa = 0.792). For the detection of the high-grade cervical intraepithelial neoplasia, the sensitivity of HC-II and real-time PCR was 96.7% and 94.6%, respectively, and the negative predictive values were 99.5% and 99.2% respectively. Conclusions Real-time PCR has good accordance with HC-II for HR-HPV DNA detection and can been used in cervical cancer screening.