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The efficient and cost-effective harvesting of food-grade Euglena gracilis remains a critical challenge in microalgal food production. This study presents an innovative, food-safe approach integrating pH preconditioning, chitosan biopolymer flocculation, and green light irradiation to leverage E. gracilis’ natural phototactic behavior. Response surface methodology optimized the parameters (pH 6.49, 46.10 mg·L−1 chitosan, and 60 min green light), achieving 93.07% biomass recovery, closely matching the predicted 92.21%. The synergistic effects of pH-modified chitosan flocculation and phototaxis significantly enhanced the harvesting efficiency compared to conventional methods. Notably, harvested cells maintained substantial photosynthetic capability, as evidenced by chlorophyll fluorescence analysis, ensuring the preservation of nutritional quality. Economic analysis revealed exceptional harvesting cost-effectiveness at 2.35 USD per kg of dry weight biomass harvested. The method’s use of food-grade chitosan and non-invasive light stimulation ensures product safety while minimizing the environmental impact. This sustainable and economical approach offers a promising solution for industrial-scale production of food-grade E. gracilis while demonstrating potential applicability to other phototactic microalgae species.