The escalating misuse of antibiotics has precipitated a dramatic rise in bacterial drug resistance, rendering conventional therapies increasingly ineffective. In this post-antibiotic era, phage lysins have emerged as a novel class of antimicrobial agents, attracting significant attention for their precise targeting of drug-resistant pathogens. Despite promising preclinical results - highlighting their rapid bactericidal efficacy and synergistic potential with other antimicrobials - the clinical translation of lysins remains limited by unresolved challenges related to efficacy, safety, pharmacokinetics, and dosing optimization. This review provides a comprehensive overview of lysin research advancements, discusses diverse application strategies, and critically evaluates therapeutic outcomes from animal models and early clinical trials. Additionally, it addresses the key obstacles impeding lysin development and proposes practical solutions and future research directions to unlock the full clinical potential of this innovative antimicrobial strategy.