A method for detection of sulfamethazine(SMT)in milk by coupling indirect inhibition assay with a self-assembled monolayer was developed. The method of immobilization of SMT-BSA on the chip surface was also introuduced. A series of milk samples with spiked 0, 5, 10, 20, 30, 40 ng/mL of SMT were analyzed by the SPR biosensor and the standard calibration curve of SMT in milk was constructed. The intra-assay relative standard deviation (RSD) and recovery were 6.3% and 102.8%, respectively. The inter-assay deviation (7 days) was less than 6 IU, and demonstrated the method’s stability. The high stability of the SPR sensor underlies the potential of the SPR method as a stable and high-precision tool for detection of SMT.