Glyphosate is widely used as a common pesticide. However, glyphosate residues in food is harmful to human health. In this paper, we proposed a flexible SERS sensor of PANI/GO/Ag film for detecting the pesticide residue of glyphosate. The Raman peak at 1045cm -1 of glyphosate was selected as the object to evaluate the SERS ability. To study the Raman enhancement ability of the flexible SERS sensor on glyphosate, the PANI/GO/Ag film was soaked in different concentrations of glyphosate solution to test the characteristic peak value. The results showed that the EF of glyphosate molecules was 1.1×10 6 and the LOD was 2.34 nM. After 15 days of storage at room temperature, the SERS enhanced performance of the sensor was up 92% of the initial strength. The PANI/GO/Ag flexible SERS sensor proposed in this paper has the advantages of high efficiency, rapid and specific recognition, which can be widely applied to the current agriculture, biomedicine, food industry, and other fields.