In this work, 5 nm Au film was deposited on 100 nm polystyrene (PS) nanobeads and tuned by three different temperatures of a short rapid thermal annealing (RTA) process, 250°, 500° and 750°. Results show that structural alteration of Au-film morphology due to RTA led to a gradual enhancement of SERS response on the detection of Rhodamine 6G (R6G) Raman probe achieving the highest enhancement factor (EF) at 1.08 × 108 by 750° of RTA. Overall, a novel approach of a non-lithographic SERS substrate with tunable plasmonic response assisted by RTA was achieved.