Interleukin-12 (IL-12) is a promising pro-inflammatory cytokine for cancer immunotherapy, but its toxicity and short half-life in serum limit its clinical application. Tumor-targeted delivery of IL-12 by fusion with either antibody or secretion by chimeric antigen receptor T (CAR-T) cells showed reduced systematic toxicity; however, the poor tumor microenvironment (TME) response or the lack of systematic IL-12 regulation still remains risk of low efficacy or high toxicity. Here, we developed TME-specific delivery of IL-12 by a tumor-targeted adeno-associated virus 9 (tAAV9). The tAAV9 was formed by an anti-folate receptor 1 (anti-FOLR1) antibody fragment conjugated with AAV9 via highly efficient Spy-ligation. With targeted infection of FOLR1+ cells in vivo, intravenous (i.v.) administration of tAAV9 specifically delivered IL-12 (tAAV9-IL-12) to TME and significantly suppressed tumor progression with favorable safety profile compared with rAAV9 (recombinant wild-type AAV9) delivery. Moreover, the IL-12 level in the serum was decreased significantly with the suppression of tAAV9-IL-12-infected tumor cell, so that generates promising negative feedback to ensure the safety profile.