Objective: This study provided a new research direction for the detection of ofloxacin content. Methods: Using biomass reduction method to formulate a reduced graphene oxide-gold composite nanomaterials (rGO-AuNPs) , which had high surface enhanced Raman scattering (SERS) activity. The successful synthesis of rGO-AuNPs was demonstrated by ultraviolet visible spectroscopy, transmission electron microscopy and scanning electron microscopy, respectively. The Raman enhancement factor (EF) of rGO-AuNPs was measured and calculated. Based on the SERS hot spot effect and the specific binding ability of nucleic acid aptamer, ofloxacin content detection system was established, the detection conditions were optimized, the standard curve was established, and apply it to OFL detection in pond water sample. Results: rGO-AuNPs composite nanomaterials was successfully synthesized by using Lilium casa blanca petals biomass as reductants. Gold nanoparticles were observed on reduced graphene oxide sheets and the EF was 3.88×10⁷. Additionally, SERS intensity had a good linear relationship with ofloxacin mass concentration in the range of 1 to 500 ng/mL, and the limit of detection was 0.3 ng/mL. The recovery was 96.28% to 102.84%, and the relative standard deviation was less than 10%. Conclusion: The synthesized rGO-AuNPs had high SERS activity and a positive application prospects in ofloxacin detection.