Protein is an essential nutrient for humans to sustain life, but it is predicted that it will be challenging to secure protein through the traditional livestock industry in the future. Microalgae has high future value as an alternative protein food source due to resource utilization and sustainability advantages. In order to increase productivity, the culture conditions of microalgae, Chlorella vulgaris, Dunaliella salina, and Scenedesmus obliquus were examined in this study. The optimal culture conditions of C. vulgaris were mixotrophic culture, 25oC culture temperature, 7.0 initial pH, 10% initial inoculation, stirring culture, 3000 Lux light intensity, and 24L:0D light/dark cycle period with red LED. For D. salina, the optimal culture conditions were mixotrophic culture, 20oC culture temperature, 8.0 initial pH, 10% initial inoculation, stirring culture, 6000 Lux light intensity, and 12L:12D light/dark cycle period with white LED. For S. obliquus, the optimal culture conditions were mixotrophic culture, 30oC culture temperature, 8.0 initial pH, 10% initial inoculation, stirring culture, 4500 Lux light intensity, and 14L:10D light/dark cycle period with fluorescent light. These findings can be used as important information for increasing the production of microalgae as an alternative protein material resource in the future.