Gas sensors based on polyaniline (PANI) materials often have some issues of intrinsic poor stability, low sensitivity, and slow response-recovery rate. To solve these issues, the combination of PANI and metal oxide nanoparticles is a common effective strategy. However, the agglomeration of these nanoparticles in the composite is still a major challenge, decreasing NH3 sensing response and stability. Herein, a flexible NH3 sensor based on PANI/Ti3C2TX/TiO2 (PMT) composite was successfully fabricated by ultrasonic spray pyrolysis and in-situ polymerization. The introduced 3D crumpled Ti3C2TX/TiO2 sphere (MT) with TiO2 nanoparticles in-situ uniform growth on the Ti3C2TX effectively prevented nanoparticles agglomeration in PMT. The optimized PMT film sensor displayed outstanding NH3 sensing performance at room temperature, including high response (2.30–10 ppm NH3), low detection limit (20 ppb), good selectivity, and excellent mechanical reliability. The superior sensing properties were attributed to the synergetic effect of unique structure of MT, the improved protonation degree of PANI component, and the formation of heterojunctions. Moreover, the detection of fish spoilage has been realized using this film sensor. The present work provides a new strategy to fabricate high-performance flexible NH3 sensors operated at room temperature, which may hold great promise for application in our daily life.