<p>Biofilm infections pose a severe threat to global public health owing to their persistent and recalcitrant nature. The physical barrier formed by the biofilm impedes the penetration of antimicrobial agents, leading to a significantly reduced efficacy of conventional antibiotics. Herein, we developed a polymeric micelle system that responds to the biofilm microenvironment to release nitric oxide (NO), which is capable of disrupting biofilms, thereby enhancing the bactericidal efficacy of antibiotics against embedded bacteria. The hydrophobic small-molecule NO donor was first conjugated to a diblock copolymer composed of N-hydroxyethyl acrylamide and N-acryloyl morpholine to yield an amphiphilic diblock copolymer. This amphiphilic copolymer then self-assembles into polymeric NO-releasing micelles (PNOM). Upon exposure to thiol-containing molecules in the reducing biofilm microenvironment, PNOM responsively released NO in a sustained manner over several days. <i>In vitro</i> studies have demonstrated that PNOM significantly potentiated the anti-biofilm efficacy of levofloxacin (Lev) against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). The combination of PNOM and Lev dispersed 85.3% of the biofilm biomass and eradicated 98.8% of the embedded bacteria. Moreover, in a murine model of implant-associated MRSA biofilm infection, PNOM was validated to enhance the antibiofilm efficacy of Lev <i>in vivo</i>, achieving a bactericidal rate of 93.9 % for MRSA biofilms and significantly alleviating inflammation. In summary, we designed a polymeric micelle system that triggers NO release in response to a thiol-rich biofilm microenvironment, thereby disrupting biofilm formation and enhancing the antibiofilm effect of antibiotics against MRSA. This approach represents a promising therapeutic strategy for treating stubborn biofilm-associated infections.</p>