Thioether-bonded bicyclic peptide can resist proteolytic degradation and become a promising scaffold for peptide-based inhibitor discovery. Chemical synthesis of thioether-bonded bicyclic peptide usually requires two orthogonal Cys protecting groups. Here, we report an efficient strategy for its preparation by using the commonly used Trt-protected and StBu-protected cysteines. Due to the thiol-disulfide exchange, S-StBu group is generally considered incompatible with sulfhydryl group under neutral conditions. We find that, in DIPEA-containing DMF solution, the peptide that contains both sulfhydryl and S-StBu groups can undergo selective thiol bisalkylation while maintaining the integrity of the S-StBu groups. In our strategy, the formation of two thioether bonds are both conducted in solution, and can be easily tracked by HPLC, which may be beneficial for the chemical synthesis of thioether-bonded bicyclic peptides.