PEGylating is an effective way for prolonging the half-time period and decreasing the immunogenicity of protein drugs. With experiments of single factor, it was proved that the optimal processes for PEGylating the fused protein of LL-37 and interferon (IFN)-α2a were:PEG molecular weight was 5 000, fused protein concentration was 0.6 mg/mL, the mole ratio of protein to mPEG5000-SS was 1:10, the reaction temperature was 4℃, and the pH was 9.0, respectively. With orthogonal experiments, we proved that the influential order of 3 main factors is:the fused protein concentration > the mole ratio of protein and mPEG5000-SS > pH and the optimal conditions were the fused protein concentration as 0.6 mg/mL, the mole ratio of protein and mPEG5000-SS as 1:10, pH as 8.8. Under these optimal conditions, the average rate of PEGylated protein with 3 times parallel experiments was 86.98%. After PEGylated, the interferon activity and antimicrobial activity of fused protein could be remained higher than 58% and 97%, respectively.