Single-atom catalysts (SACs), featuring atomically dispersed metal sites on tailored supports, hold immense potential for bridging heterogeneous catalysis and biomedical innovation, yet their intracellular application remains hindered by insufficient activity under physiological conditions and poor biological targeting. In this study, we report the design of palladium (Pd) SACs via atomic immobilization of Pd sites on a UiO-66-NH₂ metal–organic framework, denoted as PdSU, to enable spatially resolved bioorthogonal catalysis within living systems. The tailored coordination geometry and maximized active-site exposure of PdSU confer exceptional catalytic performance in bioorthogonal depropargylation reactions in both aqueous media and intracellular environments. Moreover, we demonstrate that the functionalization of PdSU with mitochondria-targeting triphenylphosphonium (TPP) and tumor-targeting biotin enables organelle-specific catalysis and cancer cell-selective prodrug activation, respectively. Notably, biotin-modified PdSU (Biotin@PdSU) drives intracellular conversion of the inert prodrug of 5-fluorouracil (Pro-5-Fu) into cytotoxic 5-fluorouracil within cancer cells, suppressing tumor growth both in vitro and in vivo. By combining atomic-level catalytic control with biologically adaptive therapeutic modulation, this study not only advances the rational design of SACs for bioorthogonal catalysis but also establishes a paradigm for spatially controlled prodrug activation.