A novel nanocomposite hydrogel-based Surface-enhanced Raman scattering (SERS) platform was successfully fabricated using Ag NPs/S-CNF/PVA hydrogel as the core matrix, demonstrating exceptional swelling-deswelling behavior. By evenly distributing a large number of Ag NPs on the surface of S-CNF, a one-dimensional tightly arranged "hot spot" was successfully constructed, which significantly improved the SERS effect and improved the sensitivity and uniformity of the SERS platform. The experimental results show that Rhodamine 6G (R6G) of 10-7 M can be detected, RSD = 1.90 %. At the same time, PVA hydrogel, as an excellent packaging material, can effectively isolate Ag NPs/S-CNF/PVA composite from the external environment, which helps to maintain the SERS property of the film, but also makes the film more durable and reliable in practical applications. By using different signal molecule modification, different substances were determined. It is worth mentioning that the substrate showed excellent SERS activity on glucose and pH, which has the potential to be applied in non-invasive tear glucose and pH monitoring system. Given its excellent flexibility and repeatability, the substrate is expected to be integrated into a variety of wearable biosensors, thus advancing the development of biological process research. Overall, by combining a flexible SERS 3D substrate, this approach shows great potential and application prospects in the study of biological processes.