Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical application. The PEGylation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEGylated hirudin were investigated. When the PEGylation reaction was conducted under 4 degrees C after 10h, in the borate buffer at pH 8.5, with the molar ratio 250 : 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro. Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.