Detection of HPV infections requires a robust time-effective single-step method for efficient screening. A molecular beacon-based one-step multiplex real-time PCR system was developed to detect 15 high-risk (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and 5 low-risk HPV types (HPV types 6, 11, 42, 43, 44). Molecular beacons detecting high-risk types are 5'-FAM-3'-DABCYL-labelled, molecular beacons for low-risk detection are 5'-TET-3'-DABCYL-labelled, while the internal control added before sample DNA extraction is detected by a 5'-FAM-TexasRed-3'-DABCYL wavelength-shifting molecular beacon. Accordingly, fluorescent data for HPV detection are collected at 530 nm for high-risk types, 560 nm in case of low-risk types and the reaction internal control is detected at 610 nm on a Roche LightCycler 2.0 instrument. The sensitivity for detected types varies between 22 and 700 copies/reaction. The clinical performance was tested on 161 clinical sample DNAs. The MB-RT PCR results were compared to the typing results obtained by the L1F/L1R PCR and hybridization-based system described previously, and the concordance rate between the two systems was 89.44%. The favorable characteristics shown by this multiplex single-step real-time HPV detection system make this promising approach worthy for further development and application for clinical screening.