Au/MoS2/polypyrrole (Au/MoS2/PPy) nanocomposite is successfully synthesized by the liquid-phase ultrasonic exfoliation process combined with in-situ polymerization method and then Au particles are decorated on its surface. Abundant sulfur (S) vacancies in the exfoliated layered MoS2 nanosheets act as active adsorption sites, and Au particles also play an efficient catalyst to enhance the NH3 gas sensing properties of Au/MoS2/PPy flexible sensor at room temperature. It results in higher NH3 gas sensing response, faster recovery rate, and more stable sensing performance even under complex environment conditions such as high relative humidity and high bending angle/numbers compared with that of pristine PPy sensor. The enhancement sensing mechanisms of Au/MoS2/PPy sensor are attributed to the p-n heterojunction between layered MoS2 and PPy, the active sites of S vacancies, the catalysis and dehydrogenation effect of Au particles, which have been verified by XPS, current-voltage (I-V), and in-situ FITR, etc. Moreover, the density-functional theory (DFT) calculations are employed to further illustrate the selectivity sensing mechanism, and reveal the S vacancies, the nanocomposite of MoS2/PPy possess high superiority for improving NH3 sensing properties at room temperature.