A novel, operational, reliable, flexible gas sensor based on silk fibroin fibers (SFFs) as a substrate was proposed for detecting the freshness of pork. Silk is one of the earliest animal fibers utilized by humans, and SFFs exposed many biological micromolecules on the surface. Thus, the gas sensor was fabricated through polyaniline (PANI) and silver nanowires (AgNWs) and deposited on SFFs by in-suit polymerization. With trimethylamine (TMA) as a model gas, the sensing properties of the PANI/AgNWs/silk composites were examined at room temperature, and the linear correlativity was very prominent between these sensing measures and the TMA measures in the range of 3.33 μg/L–1200 μg/L. When the pork sample is detected by the sensor, it can be classified into fresh or stale pork with the total volatile basic nitrogen (TVB-N) as an index. The result indicated that the gas sensor was effective and showed great potential for applications to detect the freshness of pork.