Abstract: In this research, the effects of culture methods on the growth, fatty acids and amino acids of Euglena gracilis under four cultivation modes (autotrophy, mixotrophy, heterotrophy and photoinduction) at the physiological and biochemical levels were studied. The results were as follows: the fatty acid, MUFA and PUFA contents in E. gracilis were higher under photoinduction than under the other modes, measuring 2.69 g/100 g, 0.52 g/100 g and 1.475 g/100 g, respectively. In terms of SFA content, the modes were ordered as heterotrophy>photoinduction>autotrophy>mixotrophy, and the content in the heterotrophic group reached 1.008 g/100 g. The free amino acid contents were ranked from high to low as follows: photoinduced group, mixotrophic group, heterotrophic group and autotrophic group, measuring 381.57 mg/g, 358.1 mg/g, 330.17 mg/g and 231.1 mg/g, respectively. The mixotrophic group had the highest essential amino acid content (134.37 mg/g). The results of the study showed that the fatty acid, MUFA and PUFA contents in E. gracilis were increased by photoinduction, the saturated fatty acid content in E. gracilis was increased by heterotrophic culture, and the essential amino acid content in E. gracilis was increased significantly by mixotrophic culture (P<0.05). The results may provide a scientific basis for elucidating the response of E. gracilis to different culture methods. Furthermore, they provide theoretical support for the development and application of this species.