The invention provides a novel penicillin-binding protein Bt-PBP3 and encoding genes thereof. The protein contains an amino acid sequence shown in SEQ ID No.2 or the protein is obtained by substitution, deletion and/or addition of one or more amino acids on the amino acid sequence shown in the SEQ ID No.2 and has the equal activity. The protein can be used for preparing a penicillin detection reagent, and used for penicillin detection, no expensive instrument equipment is needed, the sample pretreatment process is simple, and the requirement for the technological level of analysis personnel is low, and the protein is suitable for rapid on-site detection.