To achieve high-efficient conversion of nitrate to protein and chlorophylls from synthetic high-nitrate wastewater by mixotrophic Chlorella pyrenoidosa, the synergetic effects of initial NaNO3 concentration and pH-set were investigated in 5 L photo-fermenter. Results showed that the highest NO3− consumption rate (5.15 g L−1 d−1) and biomass concentration (23.40 g L−1) were achieved under 10 g L−1 NaNO3 and pH 6.5 at 60 h, along with high contents of protein (51.47 % DW) and chlorophylls (4.52 % DW). This study demonstrated the synergetic strategy was superior to enhance biomass and protein production with improvement of nitrate removal by mixotrophic fermentation of C. pyrenoidosa as cell factory, showing a great potential application in high-nitrate wastewater bioremediation and resource utilization through high-efficient conversion of NO3− to high-protein and high-chlorophyll biomass.