Spray drying is a very popular method for microalgal biomass drying; however, systematic research on the response of the biochemical composition during the process of spray drying has not been addressed thus far. This study investigated the influence of the inlet temperature and the initial solid content on the biochemical composition of spray-dried Scenedesmus acuminatus biomass. The fatty acid composition and contents of CHNS, lipids, carbohydrates, protein, starch, and pigments were analyzed to characterize the quality and bioactivity of the dried product. The results showed that the moisture content of the dried microalgal powder decreased with increasing inlet temperature and initial solid content, and the lowest moisture content of 2.37%, with a higher drying yield of 84%, was achieved at an optimized inlet temperature of 220 °C and an initial solid content of 16%. The biochemical compositions of CHNS, total lipids, carbohydrates, protein, starch, and fatty acids in the spray-dried biomass were similar to those in the freeze-dried biomass and were barely altered throughout the spray drying process. The pigment partially degraded as the inlet temperature increased; however, this degradation could be alleviated by increasing the initial solid content of the microalgal suspension because cell aggregates provided protection. Thermogravimetric analysis (TGA) further confirmed that spray drying did not affect the quality of proteins, lipids, or carbohydrates, suggesting that the spray drying technique could be applied to S. acuminatus for the production of both biofuel and nutritional supplements. These results may serve as a reference for the selection of the drying method, the utilization of the nutritional components in S. acuminatus, and the selection of biochemical parameters for spray drying performance evaluation.