As an excellent cell factory rich in polyunsaturated fatty acids, beta-1,3-glucan, amino acids, and vitamins, Euglena has gained great attention with high-value products in recent years. In order to determine the optimal cultivation method of Euglena pisciformis AEW501, the effects of phototrophic, mixotrophic, and heterotrophic mode on its growth were investigated, and humic acid (HA) as a growth stimulant was added into the mixotrophic culture system to verify the hypothesis that HA addition could accelerate the growth and lipid accumulation of E. pisciformis AEW501. The results indicated that biomass of mixotrophic mode (330 +/- 0.05 mg L-1) was 4.80 times and 7.65 times higher than that of phototrophic and heterotrophic mode, respectively, and mixotrophy was proved as the optimal trophic mode. Moreover. The maximum biomass productivity (34.63 mg L-1 day(-1)) and the highest lipid content (59.51%) were achieved in 50 mg L-1 HA treatment though there was no significant difference between 10 mg L-1 and 50 mg L-1 treatments. While photosynthetic efficiency of photosystem II, pigments content, cell size, and yield of eicosapentaenoic (EPA) and docosahexaenoic (DHA) in 10 mg L-1 HA treatment were the highest. It could be concluded that HA showed good promotion effects on growth and high-value products accumulation of E. pisciformis AEW501. This research will be helpful for the large-scale cultivation of E. pisciformis AEW501.