In the present paper, silver colloid films prepared by self-assembly method were used as surface-enhanced Raman scattering(SERS) active substrates to achieve trace detection of antibiotics in water. Silver colloids were prepared using the microwave heating method, and silver colloids films were prepared with self-assembly method. The enhancement of sliver colloid films to antibiotics was analysed by changing the pH value of silver colloid and times of films. Significant effects of pH value on silver colloid films were observed. And the silver colloid films with five times, prepared by silver colloids with pH 4 had the best enhancement factor. They were used as SERS active substrates to detect three antibiotics (chloramphenicol, ciprofloxacin, enrofloxacin). The experimental limits of detection were 120, 15, 120 nmol·L-1, respectively. These results show that such substrate has a very high sensitivity and application value, and might be able to be used for trace detection of antibiotics in aquiculture.