The composite of gold and PDMS hold remarkable potential for surface stress-based biosensor applicaitons. However, the gold and PDMS in composite suffers from poor compatibility with each other and suppresses the performance of the biosensor. Here, we conduct two-step synthesis of the composite of gold and PDMS by in-situ reduction and glucose reduction of HAuCl4. The composite shows stable conductive gold nanoparticle network (SC-AuNW). In in-situ reduction, Au-seeds are embedded in PDMS surface. Furthermore, AuNPs are also generated inside the PDMS and enhance conductivity. The SC-AuNW plays a crucial role in tunneling conductivity of composite film and exhibits an excellent sensitivity to surface stress. We construct SC-AuNW-PDMS biosensors based on surface stress for glucose detection with a detection limit of 0.021 mM, high repeatability and specificity. Our research can promote the application of flexible conductive films in biosensors for disease diagnosis.