This study has developed a sensitive and inhibitory surface plasmon resonance（SPR） immunosensor based on the Biotin-avidin system （BAS） for the detection of streptomycin （STR） residue in milk. Methods： STR hapten （STR-OVA） is prepared by direct cross-linking method， and coupled with biotin （BHNS） by activated ester method to obtain antigen （BNHS-STR-OVA）， the antigen is combined with streptavidin （SA） on the SA chip. Based on the principle of immune suppression， the antibody for STR at a stable concentration was simultaneously bound by the STR and the antigen BNHS-STR-OVA. According to the SPR response value obtained under different STR concentretions， the inhibition ratio was calculated and the accurete， sensitivity and celerity analysis of STR in milk could be realized according to the inhibiting ratio curve. HPLC-MS was used to test the accuracy of the method. Results： the antigen was fixed at 6 μg； the mass concentration of antibody was established at 60 mg/mL； the detection limit （IC15） and the sensitivity （IC50） for STR using the proposed SPR immunosensor achieved to 0.37 μg/L and 1.65 μg/L； the detection process could be finished within 10 min， and one chip could be reused more than 90 times. Conclusion： BAS-SPR enhanced and suppressed SPR immunosensor has offered a simple， fast， low-cost detection strategy with high sensitivity for STR， which can meet the requirements of efficient detection of STR in milk samples.