It is an effective solution to overcome the bottlenecks of commercial production of microalgal biomass by providing cost-effective and environment-friendly organic carbon sources for microalgal mixotrophic growth. In this study, effects of lignocellulose-related mannitol and xylitol on the growth, photosynthetic pigment content, cell morphology, and metabolites biosynthesis of freshwater microalga Euglena gracilis were investigated. The results revealed that both mannitol and xylitol effectively promoted the growth of E. gracilis, and at the optimal dosage of 4 g·L-1, the biomass yield was increased by 4.64-fold and 3.18-fold, respectively. Increase of cell aspect ratio was only observed in mannitol treatment groups, indicating that E. gracilis had different physiological responses to mannitol and xylitol. Fourier transform infrared spectroscopy combined with multivariate analysis was applied to analyze the cellular components. The lipid content of E. gracilis was significantly promoted by these two sugar alcohols, which would increase its potential in biofuel production.