Abstract: The penicillin binding protein gene from bacillus thuringiesis(Bt-pbp2)was cloned and expressed in the host E.coli. Protein Bt-PBP2 extracted from E.coli was confirmed to have interaction with the penicillin G using the Microscale Thermophoresis(MST)technology. The equilibrium dissociation constant between Bt-PBP2 andpenicillin G(Kdvalue),was 10.36 nmol/L.The competitive enzyme-linked method of detection and determination ofpenicillin residues in Cow Milk was established according to the interaction between protein Bt-PBP2 and penicillin.Using the method,the dose-dependent linear relationshipis evident between the absorbance(A450)and the concentration of penicillin G in the range of 4~256 μg/L.The average adding recovery rate of this methodwas above 93%±5.24%.This research was showed that protein Bt-PBP2 is capable ofdetecting the penicillin residues in cow milk,and laid the foundationsfor the highly efficient test strip for the detection of penicillin residues.