<p>Triple transfection of HEK293 cells is the most widely used method for producing recombinant adeno-associated virus (rAAV), a leading gene delivery vector for human gene therapy. Despite its tremendous success, this approach generates several vector-related impurities that could potentially compromise the safety and potency of rAAV. In this study, we introduce a method for high-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling (AAVPure^Mfg). Compared with traditional triple transfection, AAVPure^Mfg substantially improves vector purity by reducing prokaryotic DNA contaminants by 10- to 50-fold and increasing the full capsid ratio up to threefold. Mechanistically, Bxb1-mediated excision of the transgene cassette generates a minicircle cis construct devoid of bacterial sequences and ensures synchronized colocalization of trans and cis constructs in productive cells. Furthermore, we developed iterations that enhance vector genome homogeneity and streamline the production of rAAV with various transgenes, serotypes, and ITR configurations. Overall, our findings demonstrate that AAVPure^Mfg overcomes the inherent limitations associated with triple transfection, offering a broadly applicable and easy-to-implement method for producing high-purity rAAV with reduced plasmid costs.</p>