Chemotherapies for cancer treatment usually suffer from poor targeting ability and serious side-effects. To improve the treatment efficiency and reduce side effects, photoactivatable chemotherapy has been recently proposed for precise cancer treatment with high spatiotemporal resolution. However, most photoactivatable prodrugs require decoration by stoichiometric photo-cleavable groups, which are only responsive to ultraviolet irradiation and suffer from low reaction efficiency. To tackle these challenges, we herein propose a bioorthogonal photo-catalytic activation strategy with riboflavin as the catalyst for in situ transformation of prodrug dihydrochelerythrine (DHCHE) prodrug into anti-cancer drug chelerythrine (CHE), which can efficiently kill cancer cells and inhibit in vivo tumor growth under light irradiation. Meanwhile, the photo-catalytic transformation from DHCHE into CHE was in situ monitored by green-to-red fluorescence conversion, which can be used for precise control of the therapeutic dose. The photocatalytic mechanism was also fully explored by means of density functional theory (DFT) calculations. We believe this imaging-guided bioorthogonal photo-catalytic activation strategy is promising for cancer chemotherapy in clinical applications.