Polythiophene nanocomposite nanofibers (70–150 nm in diameter) containing ZnO nanoparticles were synthesized through a self-assembly process in the presence of CTAB as the surfactant. Gas-sensing properties of the prepared sample have been investigated for different concentrations of ammonia gas at room temperature. A template-like model was proposed to interpret the formation of the composite nanofibers. Gas sensing tests showed that the chemiresistor based on the as-prepared hybrid has high sensitivity, excellent repeatability, long-term stability and short response time to ammonia gas at room temperature. Therefore, the ZnO/PT hybrid can be expected to be potentially used as a gas sensor material for detection of ammonia gas at room temperature. The sensing mechanism of ZnO/PT nanocomposite to ammonia gas was presumed to be the effect of p-n heterojunction between ZnO nanoparticles and PT.