The invention discloses substrate protein for detecting Sortase A activity. The nucleotide sequence of the substrate protein is as shown in SEQ ID NO.1; and the amino acid sequence of the substrate protein is as shown in SEQ ID NO.2. The substrate protein is prepared by prokaryotic expression vector expression without chemical synthesis and modification. The preparation can be realized under general experiment conditions, cost is low, and period is short. In addition, the preparation is not limited by chemical synthesis technology. Success rate for preparation of the substrate protein is high. The substrate protein has a good application prospect. The invention also discloses a substrate protein expression vector for detection of Sortase A activity and a construction method thereof and also discloses a method for detecting Sortase A activity of staphylococcus aureus by the utilization of the above substrate protein expression vector.