Trace detection of antibiotic ciprofloxacin by surface-enhanced Raman scattering (SERS) remains a challenge, because this compound is water-insoluble and amphoteric and it has no functional groups to combine with gold nanoparticles. Herein, ciprofloxacin was physically adsorbed on a self-assembly gold nanofilm for realizing quantitative SERS detection of ciprofloxacin in fish. A gold nanofilm containing ciprofloxacin spontaneously formed at the interface of gold colloids/n-hexane by vortex mixing in a few seconds without ethanol or other organic additives. The density of the gold nanoparticles on the interface which influences the SERS effect was controlled by varying the vortex mixing time and the volume of n-hexane. Furthermore, the proposed substrates with an enhancement factor of 2.7 × 10⁶ were successfully applied for the determination of ciprofloxacin in fish samples with a linear range from 0.5 to 10.0 μg/mL and a detection limit of 0.19 μg/mL. The recovery measurements showed that the proposed SERS method had satisfactory precision and accuracy.