Abstract: The aim of the study was to improve the yield of rapeseed meal protein and reduce the content of glucosinolate by optimizing the alkali-extraction and acid-precipitation process, and the bioactivities were also explored. And single factor experiment and response surface analysis were used. The results showed that the optimal conditions were as follows, solid-liquid ratio 1:40 g/mL, pH13.1, temperature 46 ℃ and extraction time 82 min. Under these conditions, the average yield of protein and glucosinolate content were 8.78% and 1.65%. And then pH4.0 was used for protein precipitation, while the sediments and supernatant were processed by vacuum freeze-drying and spray-drying respectively and correspondingly two sorts of protein extractions, rapeseed meal precipitated protein and rapeseed meal acid soluble protein. Furthermore, SDS-PAGE analysis showed that their main components were cruciferin (Mw 31 kDa、14~22 kDa) and napin (Mw<14.4 kDa), respectively. And the crude protein of rapeseed meal precipitated protein (78.10%±0.53%). What’s more, both two protein extraction processed the ability of scavenging ABTS free radicals and inhibiting effects on S. aureus, and the EC₅₀ of the two extractions were 1.372 mg/mL and 0.438 mg/mL. Overall, this study might provide a theoretical basis for expending the application of rapeseed meal protein in the future.