This study explored the sustainable valorization of fruit waste extracts from sugarcane bagasse (SB), banana peel (BP), and watermelon rind (WR) for Euglena gracilis biomass and β-1,3-glucan production. The extracts were prepared using water extraction (WE), high-temperature and pressure treatment (HTP), and dilute sulfuric acid treatment (DSA). The DSA-treated extracts consistently yielded the best results. E. gracilis cultured in SB-DSA showed the highest cell density with a 2.08-fold increase compared to the commercial HUT medium, followed by BP-DSA (1.35-fold) and WR-DSA (1.70-fold). Photosynthetic pigment production increased significantly, with chlorophyll a yield being highest in SB-DSA (1.90-fold increase). The chlorophyll a/b ratio and total carotenoid content also improved, indicating enhanced light-harvesting capacity and photoprotection. Photosynthetic efficiency, measured by chlorophyll fluorescence, notably improved. The maximum quantum yield of PSII (Fv/Fm) increased by up to 25.88% in SB-DSA, suggesting reduced stress and improved overall photosynthetic health. The potential photochemical efficiency (Fv/F0) showed even greater improvements: up to 40.53% in SB-DSA. Cell morphology analysis revealed larger cell aspect ratios, implying a more active cellular physiological state. β-1,3-glucan yield also increased by 23.99%, 12.92%, and 23.38% in SB-DSA, BP-DSA, and WR-DSA, respectively. This study demonstrates the potential of pretreated fruit waste as a cost-effective and sustainable medium for E. gracilis cultivation, offering the dual benefits of waste valorization and high-value compound production. These findings contribute to the development of more efficient biorefinery processes and align with the circular economy principles in food biotechnology.