Suicide gene therapy holds great promise for cancer treatment, exhibiting potent efficacy across diverse tumor types. However, its clinical application remains hampered by insufficient tumor selectivity and systemic toxicity. Here, we developed tumor-specific suicide gene nanomedicines that selectively eliminate cancer cells while sparing healthy tissues. These nanomedicines comprise lipid-assisted polymeric nanoparticles encapsulating plasmids driven by tumor-specific promoters, enabling selective expression of a mutant herpes simplex virus thymidine kinase (SR39TK) in tumor cells. The expressed SR39TK converts the prodrug ganciclovir (GCV) into its cytotoxic triphosphate form (GCV-PPP), inducing tumor-specific apoptosis. Notably, the tyrosinase promoter-driven NP_Tyr-SR39TK enables melanoma-specific expression and strong antitumor efficacy, whereas replacing the tyrosinase promoter with a survivin promoter yields NP_Sur-SR39TK, which extends this precision cytotoxicity to other tumor types while maintaining safety in normal tissues. Overall, this study introduces a versatile and tumor-selective gene therapy strategy, offering a promising avenue for advancing suicide gene therapy.