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The present study has focused on the effects of hypericin (Hyp) based photodynamic inactivation (PDI) of Escherichia coli (E. coli). To evaluate the efficiency of Hyp based PDI of E. coli, single factor experiments and response surface optimization experiment were conducted to obtain the optimum parameter values (36 µM Hyp, 5.9 J cm−2 light dose: 16.4 mW cm−2, 60 W, 260 s, 590 nm and 68 min incubation time) and finally achieved a 4.1 log CFU mL−1 decrease of E. coli. Cell-Hyp interaction and intracellular reactive oxygen species (ROS) level were detected by fluorescence spectrometric photometer. Data indicated that Hyp possessed a strong ability to bind with cells. In addition, a significant increase was observed in intracellular ROS level after Hyp-based photosensitization treatment. Therefore, Hyp-based photosensitization seems to be a promising method to efficiently inactivate E. coli. It is expected to be a safe, efficient, low cost and practical method which can be applied in the field of food safety.