Ag/rGO nanocomposite SERS substrates are widely used for the detection of contaminations in water and food adulterants. In this work, we prosed a novel green route to synthesize Ag/rGO nanocomposite for SERS-based detection of methylene blue (MB). Ag/rGO nanocomposite is synthesized by simultaneous reduction of AgNO3 and GO with the leaf extract of Jasmine fluminense. Further, the flexible SERS substrate is fabricated by directly coating the synthesized Ag/rGO nanocomposite on cotton fabric and used for SERS-based detection of MB. The SERS studies on MB show a linear response of 0.97 over a wide detection range of 10− 3 to 10− 9 M with a minimum detection limit of 10− 9 M. The enhancement factor for MB at a concentration of 10− 9 M is 5.33 × 105 with a high signal-to-noise ratio of 31.69. This study emphasizes the potential of green synthesized Ag/rGO embedded flexible cotton SERS substrate for the traceable fingerprint detection of MB.