The alkali dissolution method was used to treat the tobacco, and the acid precipitation method was used to precipitate soluble protein. The protein extraction conditions were optimized based on the extraction rate of soluble proteins, and the antioxidant activity of tobacco soluble proteins was determined. The experimental results showed that, through the single factor test of material-liquid ratio, extraction liquid, pH, and extraction time, the center point parameters of the response surface test were determined, and the response surface optimization was carried out on this basis. The optimal process conditions for protein extraction were as follows: 0.05 mol/L phosphate buffer added 1.1% PVP, material-liquid ratio 1∶10.5（g/mL）, pH value 8.6, and the extraction rate of tobacco soluble protein is 84.95%. Tobacco soluble proteins exhibited ABTS₊· and DPPH· free radical scavenging abilities, with maximum scavenging rates observed at a protein concentration of 1.0 mg/mL, which were 58.57% and 45.97%, respectively. The extraction rate of tobacco soluble protein optimized by response surface method is high, which clarifies the antioxidant activity of tobacco soluble protein, and provides a theoretical basis for the multi-purpose development and utilization of tobacco protein.