In order to improve the phycobiliprotein production in unicellular red algae Galdieria sulphuraria under mixotrophic conditions, systematic optimization of environmental factors including the culture temperature, C/N ratio, initial cell density and light intensity were performed by using cell density and PBP content as the evaluation indices. The results showed that moderate reduction of culture temperature and C/N ratio of medium was beneficial for improving the accumulation of phycobiliprotein. Increasing the light intensity and initial cell density could promote the cell growth and phycobiliprotein synthesis. The study indicated that the optimal conditions for cell mixotrophic culture were determined as 30 ℃, C/N ratio of 6, initial cell density of 1×108 cells/mL, light intensity of 110 μmol/(m2·s). In the optimized culture system, the maximum cell density (5.48×108 cells/mL) could be achieved after mixotrophic cultivation for 8 days, leading to the highest biomass yield of 11.65 g/L which was 1.46 times that before optimization. The content of phycobiliprotein was 13.63% of dry cell weight, with the corresponding yield of 1588.36 mg/L which was 187.91% higher than that before optimization. Therefore, the systematical optimization of mixotrophic culture conditions was the effective strategy to enhance the biomass and phycobiliprotein production in G. sulphuraria.