Gene therapy has emerged as a powerful approach for treating diverse diseases, including genetic disorders, retinal diseases, and certain cancers. Real-time, noninvasive tracking of gene expression is essential for evaluating therapeutic efficacy. β-galactosidase (β-gal), a hydrolase encoded by the gene or the human gene, is widely used as a reporter of gene expression. In humans, β-gal deficiency underlies several fatal neurodegenerative disorders, including GM1 gangliosidosis. Here, we report the development of a β-gal-activated, human serum albumin (HSA)-binding gadolinium(III)-based MR contrast agent for noninvasive assessment of adeno-associated virus (AAV) gene therapy in GM1 gangliosidosis mice. The probe exhibited a gradual increase in MR relaxation rate upon incubation with β-gal in the presence of 4.5% HSA. Following intravenous administration, AAV-treated GM1 mice demonstrated distinct MR signal enhancement and kinetic profiles compared to untreated β-gal-deficient controls. This study establishes an enzyme-activated, protein-binding MR imaging strategy for real-time, noninvasive monitoring of AAV gene therapy.