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<i>Euglena</i>, a flagellated unicellular protist, has recently received widespread attention for various high-value metabolites, especially paramylon, which was only found in Euglenophyta. The limited species and low biomass of <i>Euglena</i> has impeded paramylon exploitation and utilization. This study established an optimal cultivation method of <i>Euglena pisciformis</i> AEW501 for paramylon production under mixotrophic cultivation. The results showed that the optimum mixotrophic conditions were 20 °C, pH 7.0, and 63 μmol photons m⁻²∙s⁻¹, and the concentrations of sodium acetate and diammonium hydrogen phosphate were 0.98 g L⁻¹ and 0.79 g L⁻¹, respectively. The maximal biomass and paramylon content were 0.72 g L⁻¹ and 71.39% of dry weight. The algal powder contained more than 16 amino acids, 6 vitamins, and 10 unsaturated fatty acids under the optimal cultivation. <i>E. pisciformis</i> paramylon was pure β-1,3-glucan-type polysaccharide (the purity was up to 99.13 ± 0.61%) composed of linear glucose chains linked together by β-1,3-glycosidic bonds. These findings present a valuable basis for the industrial exploitation of paramylon with <i>E. pisciformis</i> AEW501.