Adeno-associated virus (AAV)-mediated gene therapy represents a promising approach for treating genetic disorders. However, challenges remain in achieving stable transgene expression and mitigating liver injury during long-term therapy. Previous studies have implicated the activation of RIG-I-like receptors (RLRs), which detect double-stranded RNA (dsRNA), as a potential inhibitor of transgene expression. In this study, we investigated the role of the RLR pathway in AAV-transduced cells, with a focus on the generation of sense and antisense RNA, as well as the formation of dsRNA. Our findings revealed that dsRNA is produced following AAV transduction, leading to the activation of the RLR pathway and the induction of innate immune responses. Prolonged AAV transduction in mice resulted in significant liver injury, which was independent of adaptive immune activation. Instead, mitochondrial antiviral signaling protein (MAVS) activation emerged as a critical mediator of these effects. Notably, downregulation of MAVS enhanced transgene expression, suggesting that modulating MAVS could enhance the efficacy of AAV-based gene therapy. This study elucidates the mechanisms underlying dsRNA formation and RLR pathway activation, highlighting their impact on the efficacy of AAV gene therapy. These findings suggest that strategies aimed at minimizing dsRNA production and targeting the RLR-MAVS pathway could reduce immune activation and enhance therapeutic transgene expression, thereby optimizing AAV-based interventions for genetic disorders.