Objective To establish a quantitative detection method for the quantitative determination of the viable bacteria number of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk, by combining propidium monoazide (PMA) with real-time fluorescence quantitative polymerase chain reaction (qPCR). Methods Firstly, the specific primer probe of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was designed and verified, the standard curve was established using standard strains, and the reaction conditions of the PMA were optimized. Secondly, the quantities of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in the fermented milk samples were quantitatively detected. Results The optimal mass concentration of damaged bacteria of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was 10 μg/mL, and the optimal exposure time was 10 min. The PMA-qPCR detection method could accurately detect Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk. Conclusion In this study, a rapid and accurate PMA-qPCR detection method is established for the detection of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk system, which provides certain reference significance for the quantitative detection of probiotics in fermented milk products.