In order to obtain a more stable PEGylated interferon a-2b, and prolong its half life, interferon a-2b(IFN a-2b) was modified with monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD) 20000. It was found that the optimized reaction condition for the maximum bioactivity and highest PEGylation degree of the mono PEGylated interferon a-2b was as follows: in 20 mmol/L, pH 6.5, citric acid and sodium dihydrogen phosphate buffer, the concentration of IFN a-2b was 4 mg/mL, and the molar ratio of PEG/IFN a-2b was 8:1, and the reaction time was 20 h at 4oC. Under the optimized reaction condition, the mono PEGylation degree reached to 55%. Ion exchange chromatography was used to separate and purify mono PEGylated interferon a-2b from the reaction mixture. The purity of mono PEGylated interferon a-2b was higher than 97% characterized by HPLC. The bioactivity of the mono PEGylated interferon a-2b was 13.4% of the native IFN a-2b, while its half life in SD rat is much longer than the native IFN a-2b. The mono PEGylated interferon a-2b is also stable in aqueous.