Adeno-associated viruses (AAV) are among the most actively investigated vectors for gene therapy. The supply of early clinical studies with frozen drug products (DP) can accelerate timelines and minimize degradation risks. In the long term, logistical challenges of frozen DP may limit patient access. Aiming to evaluate the feasibility of a lyophilization dosage form for AAV gene therapy, the impact of buffer systems, crystalline and amorphous excipients, and moisture content on AAV stability during the lyophilization process were evaluated, and the critical factors for maintaining AAV stability were identified. More importantly, we demonstrated the potential of a lyophilization formulation for AAV long-term storage under refrigerated storage conditions.