Biocompatible and proteolysis-resistant poly-β-peptides have broad applications and are dominantly synthesized via the harsh and water sensitive ring-opening polymerization of β-lactams in the glovebox or using a Schlenk line, catalyzed by strong base LiN(SiMe 3 ) 2 . We developed a controllable and water insensitive ring opening polymerization of β-amino acid N-thiocarboxyanhydrides (β-NTAs) that can be operated in open vessels to prepare poly-β-peptides in high yields, with diverse functional groups, variable chain length, narrow dispersity and defined architecture. These merits imply wide applications of β-NTA polymerization and resulting poly-β-peptides, which is validated by the find of a HDP mimicking poly-β-peptide with potent antimicrobial activities. The living β-NTA polymerization enables the controllable synthesis of random, block copolymers and easy tuning of both terminal groups of polypeptides, which facilitated unravelling the antibacterial mechanism using the fluorophore-labelled poly-β-peptide.