A robust, efficient, and orthogonal click chemistry (copper (I)-catalyzed alkyne-azide cycloaddition) was used to prepare an antimicrobial polymer and precisely control the conjugation ratio of antibiotic molecules to polymer. Antimicrobial polyurethanes with pendant benzisothiazolinone (PU-BIT) were synthesized using click chemistry to connect azide functional polyurethane (PU-N3) and alkyne functional benzisothiazolinone (BIT-Al). The direct contact-killing and non-leaching antimicrobial properties of PU-BIT were verified by both antimicrobial drop and disk diffusion. This approach provides a new methodology and platform for the development of contact-killing and non-leaching antimicrobial materials for a variety of practical applications. This research is the first to demonstrate that the broad-spectrum BIT antibiotic is a selective antibiotic for Gram-positive bacteria when covalently linked to a polymer. PU-BIT film containing 0.8 wt% BIT exhibited a selective antimicrobial performance with bactericidal efficacy of 91.6% and 30% against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, respectively. The mechanism of the selective antimicrobial activity of PU-BIT is also discussed.