Although studies on algae-mediated photodegradation for antibiotic removal in exploring cleaner environmental systems have been reported, the specific mechanism of algal organic matter for improving antibiotic photodegradation remains unclear. Herein, we proposed the enhanced efficiency mechanism of extracellular organic matter (EOM) and intracellular organic matter (IOM) in Microcystis aeruginosa for promoting tetracycline (TC) photodegradation. The degradation efficiencies of EOM and IOM on TC were found to obtain 74.5 and 83.3%, respectively. Three-dimensional excitation emission matrix fluorescence spectroscopy combined with parallel factor analysis and Fourier transform infrared confirmed that the carbonyl compounds exhibited a significant contribution to the photosensitivity of EOM and IOM. Mechanistic investigation showed that 3EOM* and 3IOM* were identified as the principal active species in the photodegradation of TC. The EOM and IOM showed excellent detoxification ability for TC removal and remarkable tolerance in real water backgrounds, highlighting strong potential for applications. This study refined the photosensitization mechanism of algal organic matter degradation antibiotics, and offered methods and theoretical basis for environment-friendly and sustainable control of antibiotic pollutants.