Rapid and sensitive detection of contaminants in foods is crucial for minimizing potential health hazards to humans. In this work, we developed a SERS substrate fabricated by femtosecond laser induced plasma assisted ablation (fs LIPAA) for food contaminants detection. By tuning laser fluence, the distribution and size of silicon nanoparticles generated by fs LIPAA are studied. Hotspots with high uniformity can be created for SERS functionalization by decorating the substrate with a layer of silver film. It is found that the SERS performance is well optimized at a laser fluence of 5 J/cm2. The proposed Ag NPs/glass SERS substrate shows good uniformity with an enhancement factor of 2 × 105 via R6G Raman probe as compared to a traditional smooth Ag film. The detection limits of melamine in milk liquid and ciprofloxacin in water are both 1 ppm. The results demonstrate great potential to pave a new way to realize the use of SERS substrates in food safety.