Abstract: Objective: To establish a rapid detection method for viable Penicillium expansum by propidium monoazide (PMA) combined with quantitative real-time polymerase chain reaction (qPCR). Methods: PMA-qPCR detection method for viable P. expansum was established, including the optimization of the treatment concentration, dark incubation and exposure time of PMA, the screening of specific primers of P. expansum, and the conduction of qPCR. In addition, the standard curve was constructed, which was applied to the detection of artificially contaminated apples samples. The reliability of this method was also evaluated by comparing with the plate counting method. Results: The optimal PMA treatment conditions were: 10 µg/mL for PMA concentration, 5 min for the dark incubation and 10 min for the exposure time. Among the 4 pairs of primers, Pexp-patF showed strong specificity for P. expansum, which could be used as a optimal primer for PMA-qPCR detection. The correlation coefficient of the established quantitative standard curve was 0.9948, and the detection limit of the method was 10^2.6 CFU/mL. There was no obvious difference between the detection result of this method and the plate counting method, and the viable P. expansum could be detected in the non-rotted part of apples. Conclusion: The established PMA-qPCR technique could be applied to the detection of P. expansum in apples, which would provide technical support for the prevention and control of P. expansum.