NH3 is widely existed in the environment and is closely associated with various health issues. Additionally, detecting the small amounts of NH3 exhaled by patients with liver and kidney diseases offers potential opportunities for painless early disease diagnosis. Therefore, there is an urgent need for a convenient, rapid, and highly sensitive real-time NH3 monitoring method. This work presents a high-performance NH3 sensor based on olfactory receptor-derived peptides (ORPs) on a pyramid silicon nanowires (SiNWs) structure substrate. First, we successfully fabricated the pyramid-SiNWs structure on a silicon substrate using a chemical etching method. Subsequently, by dehydrative condensation reaction between the amino groups on APTES and the carboxyl groups of ORPs, ORPs were successfully immobilized onto the pyramid-SiNWs structure. This methodology allows the ORPs sensor on the pyramid-SiNWs substrate to detect NH3 as low as 1 ppb, which was the reported lowest limit of detection, with a higher response rate compared to ORPs sensors on flat SiNWs substrates. The sensors also exhibit good sensitivity and stability for NH3 gas detection. The results show the feasibility and potential applications of ORPs-pyramid-SiNWs structure sensors, in the fields of food safety, disease monitoring, and environmental protection, etc.