Cellulose fiber/paper-based surface-enhanced Raman scattering (SERS) is considered as a promising food safety detection technology due to its non-toxicity, low cost, flexibility, and hygroscopicity for possible rapid on-site agricultural product contaminant detection. However, it faces the problems of poor noble metal adhesion and toxic noble metal reducing agent. In this study, a natural macromolecule—xylan was used as both a reducing agent and a stabilizing agent to prepare stable Au-Ag bimetal nanoparticles, which were anchored on the paper surface by xylans in order to fabricate a paper-based Au-Ag bimetallic SERS substrate. The results show that the SERS substrate has a high Raman enhancement performance and reproductively. The substrate can effectively detect trace pesticide, i.e., thiram, and the limit of detection is as low as 1 × 10–6 mol/L (0.24 ppm). In addition, the paper-based SERS substrate can be used for direct detection of pesticide residues on the surface of fruit. The paper-based SERS substrate developed in this study has great potential in applications for rapid food safety detection.