In this study, a room temperature operable and selective ammonia (NH3) gas sensor based on polyaniline (PANI) nanocomposite was successfully developed on the interdigital electrode through a chemical oxidative polymerization process. This work reports the first instance of PANI being doped with amino acid (Serine) as a protonic acid, while PANI was also doped with nitric acid (HNO3) and tartaric acid (Tartaric-acid) for the control experiment. The composition of materials, surface morphology, and structural elucidation of PANI@Serine were systematically analyzed using X-ray photoelectron spectroscopy, field emission scanning electron microscopy, and X-ray diffraction, respectively. The gas sensing performances of the PANI@Serine sensor were thoroughly studied and compared with PANI@HNO3 and PANI@Tartaric-acid. The PANI@Serine sensor exhibited excellent gas sensing capabilities towards NH3 at 25 ± 2 °C, with a response of 6.12 towards 16 ppm of NH3. This response was 4.29 times higher than PANI@Tartaric-acid and 5.81 times higher than PANI@HNO3 sensor. Furthermore, the PANI@Serine sensor demonstrated a low detection limit of 10 ppm NH3, favorable recovery characteristics and stability, selectivity, and response. The density functional theory (DFT) of the three doped PANI was extensively calculated, and the adsorption behavior of NH3 was analyzed. The results revealed that PANI@Serine exhibited a double adsorption effect on NH3 gas molecules, with significantly higher adsorption performance compared to PANI@HNO3 and PANI@Tartaric-acid.