Most antibiotics contain ionizable groups that undergo acid-base dissociation giving rise to diverse dissociated forms in aquatic systems depending on the pH of the system. In sunlit surface waters, photochemical transformation plays a crucial role in determining the fate of antibiotics. This study presents a comprehensive examination of the photo-transformation degradation kinetics, pathways and photoinduced toxicity of three widely detected tetracyclines (TCs): tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC). Under simulated sunlight (λ > 290 nm), their apparent photolysis followed pseudo-first-order kinetics, with rate constants significantly increasing from H2TCs0 to TCs2−. Through competition kinetic experiments and matrix calculations, it was found that the anions HTCs− or TCs2− (pH ∼ 8–10) were more reactive toward hydroxyl radicals (•OH), while TCs2− (pH ∼ 10) reacted the fastest with singlet oxygen (1O2). Considering the dissociated species, the total environmental photo-transformation half-lives of TCs were determined, revealing a strong dependence on the water pH and seasonal variation in sunlight. Generally, apparent photolysis was the dominant photochemical process, followed by 1O2 and •OH oxidation. Different transformation pathways for the three reactions were determined based on the key photoproducts identified using HPLC-MS/MS. Toxicity tests and ECOSAR software calculations confirmed that the intermediates produced by the •OH and 1O2 photo-oxidation processes were more toxic than the parent compounds. These findings significantly enhance our understanding of the complex photochemical fate and associated risks of TCs in aqueous environments.