A room-temperature high-performance ammonia (NH3) gas sensor based on hydroxyapatite (HAp) film with three-dimensional network structure was reported in this paper. The gas sensor was fabricated on the indium tin oxide glass (ITO) interdigital electrode via simple electrochemical deposition technique. The HAp film gas sensor shows good sensitivity, high reproducibility and excellent selectivity to NH3 gas under dry and relative humidity (RH) conditions at room temperature. The response and recovery time of the HAp film gas sensor to 1000 ppm NH3 gas are 23 s and 14 s under N2 (dry) condition, and are 4 s and 11 s under air (RH ≈ 30) condition, respectively. The HAp film gas sensor has no considerable deviation in the 1000 ppm NH3 gas for 3 cycles under N2 (dry), and so is air (RH ≈ 30) conditions. The HAp film gas sensor shows excellent selectivity to methanol, acetone and ethanol under N2 (dry) and air (RH ≈ 30) conditions. Above all, two sensing mechanisms of the HAp film gas sensor to NH3 gas have been also researched in this work.