To enhance the fermentation protein yield of Auxenochlorella pyrenoidosa and its amino acid quality through combined mutagenesis, the high-protein, chlorophyll-deficient mutant strain CX41, previously obtained by the research team, was used as the original strain. Combined mutagenesis was conducted using ethyl mesylate and atmospheric and room temperature plasma, followed by high-throughput screening through a microbial microdroplet culture system and 48-well plate cultivation. Subsequently, after secondary screening via 250 mL shake flask cultivation, the mutant strain Y11 was ultimately obtained. Under dark fermentation conditions at 30 ℃ and 160 r•min-1 for 96 h in basal medium containing 30 g•L-1 glucose and 3.75 g•L-1 sodium nitrate, the biomass, protein content, and yield of Y11 reached 9.05 g•L-1, 55.54 g/100 g dry weight, and 1.26 g•(L•d)-1, respectively, which were significantly higher than those of the original strain CX41, with increases of 21.15%, 25.60%, and 51.81%, respectively. The amino acid score (94.41) and total branched-chain amino acid content (6.96 g/100 g dry weight) also improved by 3.49 points and 27.35%, respectively. Moreover, compared with CX41, the lutein content (1.12 mg•g-1), yield (10.10 mg•L-1), and productivity (2.52 mg•(L•d)-1) significantly increased by 4.67%, 26.88%, and 26.63%, respectively. This study provides a theoretical basis and technical support for the breeding of high-quality microalgal strains by using combined mutagenesis and high-throughput screening techniques.