Over the past few decades, ammonia (NH3) has been commonly used in the petrochemical and semiconductor industries. Exposure to NH3 is dangerous to life, making continuous monitoring of NH3 becomes vital. Sensitive and stable ammonia gas sensors with low power consumption have gained increasing attention. This study uses a through-silicon via (TSV) technology and atomic layer deposition to fabricate a three-dimensional (3D) TSV-structured room-temperature TiO2 gas sensor. For various NH3 concentrations, the sensor response for the 3D TiO2 gas sensor increased if the NH3 concentration is increased. In terms of the stability and the reproducibility of the 3D room temperature TiO2 NH3 gas sensor at 10 ppm NH3 gas, the sensor response is about 9.35% on average, with an inaccuracy of <± 0.8 %. For various gas measurements, the sensor also exhibits good selectivity for NH3 gas. This gas sensor shows good stability, reproducibility, and selectivity with low power consumption can be used in various applications.