Interleukin-1 (IL-1) is a pivotal mediator in the pathological progression of osteoarthritis (OA), playing a central role in disease progression. However, the rapid clearance of IL-1 receptor antagonist (IL-1Ra) from the joints may hinder the efficacy of intra-articular IL-1Ra injections in reducing OA-associated pain or cartilage degradation. Sustaining sufficient levels of IL-1Ra within the joints via adeno-associated virus (AAV)-mediated gene therapy presents a promising therapeutic strategy for OA. In this study, we constructed an IL-1Ra expression cassette employing intron insertion in the coding sequence (CDS) region to enhance protein expression levels. Furthermore, we incorporated precisely targeted liver-specific microRNA (miRNA) sequences to specifically downregulate transgene expression within hepatic tissues, thereby ensuring more targeted and controlled regulation of gene expression. A rat model of OA was employed to compare the efficacy of AAV5 and AAV9 for IL-1Ra delivery at both high and low doses. It was observed that low-dose, but not high-dose, AAV9-IL-1Ra resulted in a significant reduction in joint swelling, accompanied by a decrease in the diameter of the affected area and the preservation of biomarkers associated with trabecular bone integrity. These results highlight the great potential of AAV9-IL-1Ra in osteoarthritis therapy, with the promise of achieving long-term improvement through a single intra-articular injection.