Abstract: To enhance the in vitro antioxidant performance of raffinose, this study investigated the effects of temperature, time, pH, and the ratio of modifiers on the phosphate content in raffinose as single factors. The preparation process of raffinose was optimized by response surface methodology. The structures of raffinose before and after phosphorylation modification were characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance Spectroscopy (NMR), and their in vitro antioxidant activities were studied. The results showed that the optimal preparation process of phosphorylated raffinose (P-raffinose) was as follows: pH of the reaction system was 9, the ratio of sodium tripolyphosphate to sodium trimetaphosphate was 4:3, the reaction temperature was 77 ℃, and the reaction time was 5.3 h. Under these conditions, the phosphate content in P-raffinose was 32.14%. Characterization results indicated that raffinose was successfully phosphorylated. Activity tests demonstrated that phosphorylation modification significantly improved the hydroxyl radical scavenging capacity and total antioxidant capacity of raffinose, increasing by 27.06% and 31.40% respectively compared to raffinose. This study provides theoretical basis and technical support for the application of P-raffinose in the development of new functional foods and antioxidants.