The detection of ammonia (NH3) is significant because it can seriously damage human health and atmospheric environment. However, the development of high-performance flexible NH3 gas sensors operated at room temperature is still challenging. Thus, a flexible NH3 film sensor based on poly(4-stryenesulfonate)-doped polyaniline nanoparticle (PANI:PSS)/Ti3C2Tx composite was developed by in-situ polymerization. This optimized PANI:PSS/Ti3C2Tx film displayed the high response of 0.57–1 ppm NH3, which was improved by 1.78-fold in comparison with PANI:PSS film. Besides, the response value of this film to NH3 was 1.46 times higher than to NO2, and this value was much higher than to other interfering gases (NO, toluene, ethanol, and acetone). Additionally, this film sensor possessed excellent mechanical stability and low detection limit (20 ppb) at room temperature. The abundant Schottky heterojunctions formed in PANI:PSS/Ti3C2Tx composite, the numerous -SO3H groups introduced by PSS anion chains, and the improved protonation degree of PANI component are responsible for the enhanced sensing properties. The monitoring experiment of shrimp spoilage has demonstrated the application feasibility of this film sensor. This research provides a novel and practical method for constructing high-performance polymer/MXene based gas sensors.