Gas sensors have played a critical role in healthcare, atmospheric environmental monitoring, military applications and so on. In particular, flexible sensing devices are of great interest, benefitting from flexibility and wearability. However, developing flexible gas sensors with a high sensitivity, great stability and workability is still challenging. In this work, multi-walled carbon nanotubes (MWCNTs) were grown on polydimethylsiloxane (PDMS) films, which were further modified with polyaniline (PANI) using a simple chemical oxidation synthesis. The superior flexibility of the PANI-MWCNTs/PDMS film enabled a stable initial resistance value, even under bending conditions. The flexible sensor showed excellent NH3 sensing performances, including a high response (11.8 ± 0.2 for 40 ppm of NH3) and a low limit of detection (10 ppb) at room temperature. Moreover, the effect of a humid environment on the NH3 sensing performances was investigated. The results show that the response of the sensor is enhanced under high humidity conditions because water molecules can promote the adsorption of NH3 on the PANI-MWCNTs/PDMS films. In addition, the PANI-MWCNTs/PDMS film sensor had the abilities of detecting NH3 in the simulated breath of patients with kidney disease and the freshness of shrimp. These above results reveal the potential application of the PANI-MWCNTs/PDMS sensor for monitoring NH3 in human breath and food.