Enteric bacterial pathogens in sewage sludge easily become viable but nonculturable (VBNC) during anaerobic digestion, which escape detection by standard culture methods and pose a potential health risk. In this study, a method that is combining the standard culture method with the reverse transcription quantitative PCR (RT-qPCR) assay was developed for the quantification of bacterial pathogens in the VBNC state. The cycle threshold (C-T) values from RT-qPCR assays were linear to the bacterial number in the range from 10(9) to 10(2) most probable number (MPN) per reaction for Escherichia coli (R-2=0.9964) and Salmonella typhimurium (R-2-00.9938) and from 10(9) to 10(4) MPN per reaction for Shigella flexneri (R-2-00.997), respectively. Mesophilic anaerobic digestion (MAD) caused the bacterial pathogens in sewage sludge entering into VBNC state with the incidence indexes of 0.01-1.12 for E. coli, 2.48-436.52 for S. typhimurium, and 4.17-6.61 for S. flexneri, respectively. Given different VBNC incidence indexes of bacterial pathogens in sewage sludge by MAD, the quantification results of VBNC pathogens using RT-qPCR could provide an improved evaluation of pathogen inactivation efficiency and biological safety in sludge anaerobic digestion.