Dunaliella sp. is a prospective green microalga that can utilize both organic and inorganic carbon simultaneously. This study was aimed to determine the influence of various glucose concentrations on biomass concentration, β-carotene, and protein content of Dunaliella sp. under mixotrophic cultivation. Different glucose supplementation of 0.05 g/L, 0.10 g/L, 0.15 g/L, and 0.20 g/L were applied mixotrophically. The culture condition of Dunaliella sp. was also performed under photoautotrophic cultivation. The results exhibited that glucose administration significantly influenced the growth, biomass concentration, β-carotene, and protein production of Dunaliella sp. (P < 0.05). Supplementation of glucose in the mixotrophic culture remarkably improved cell growth, biomass production, β-carotene, and protein content of Dunaliella sp. compared to photoautotrophic culture. Increasing glucose concentration from 0.05 to 0.15 g/L increased biomass yield, β-carotene, and protein content of Dunaliella sp. The maximum specific growth rate and biomass concentration were produced at the glucose administration of 0.15 g/L with a value of 1.058 per day and 0.896 g/L, respectively. Moreover, supplementation of 0.15 g/L glucose resulted in the highest β-carotene and protein content. The results also noted that nitrate and phosphate consumption was highly related to biomass, β-carotene, and protein content of Dunaliella sp. In conclusion, the supplementation of glucose under mixotrophic conditions could improve the biomass, β-carotene, and protein content of Dunaliella sp. and could be practically used in mass-scale production. © 2021 Friends Science Publishers