Microplastics (MPs) provide a specific habitat for bacterial adhesion and the adsorption of extracellular DNA (eDNA), making them hotspots for the transformation of antibiotic resistance genes (ARGs) in the environment. However, the heterogeneous mechanism of ARG regulation by quorum sensing (QS) in MP biofilms remains unclear. This study demonstrated that QS signals, including C8-HSL, 3-oxo-C10-HSL, and C12-HSL, were detected at ng/L levels in Bacillus subtilis biofilms colonized on MPs. The addition of these three AHLs enhanced natural transformation by 1–2 orders of magnitude in both MP and natural substrate (NS) biofilms, while QS inhibitors decreased transformation frequencies by 2 orders of magnitude. Moreover, transformation frequencies in MP biofilms increased 6.0-fold under QS regulation mediated by AHLs, compared to a 2.5-fold increase in NS biofilms. QS-regulated biofilm formation and EPS secretion were found to be responsible for ARG dissemination in MP biofilms, as indicated by the positive correlation between transformation frequencies and EPS variation. Additionally, mRNA expression analysis revealed that activation and inhibition of the QS system in MP biofilms regulated the expression of natural transformation-related genes, including epsG (regulating EPS secretion) and recO (regulating eDNA transformation). These results highlight the significant influence of the QS system on ARG dissemination through heterogeneous biofilm formation and EPS secretion in MP biofilms. Understanding the effect and mechanism of QS on ARG natural transformation provides new insights into controlling ARG spread by targeting QS in host bacteria within MP biofilms.