Cancer therapies usually suffer from poor targeting ability and serious side effects. Photoactivatable cancer therapy has the significant advantage of a high spatiotemporal resolution, but most photoactivatable prodrugs require decoration with stoichiometric photocleavable groups, which are only responsive to ultraviolet irradiation and suffer from low reaction efficiency. To tackle these challenges, we herein propose a photoactivation strategy with biogenic riboflavin as the photosensitizer to promote the in situ transformation of noncytotoxic dihydroalkaloid prodrugs dihydrochelerythrine (DHCHE), dihydrosanguinarine (DHSAN), and dihydronitidine (DHNIT) into anticancer alkaloid drugs chelerythrine (CHE), sanguinarine (SAN), and nitidine (NIT), respectively, which can efficiently kill cancer cells and inhibit in vivo tumor growth. Meanwhile, the photoactivatable transformation can be in situ monitored by green-to-red fluorescence conversion, which will contribute to easy controlling of the therapeutic dose. The proposed photoactivatable transformation mechanism was also explored by density functional theory (DFT) calculations. We believe this riboflavin-promoted and imaging-guided photoactivation strategy is promising for precise cancer therapy.