Hirudin, the most potent inhibitor of thrombin found in nature, has a short half-life in serum, which significantly limits its clinical application as an anticoagulant. Recently, PEGylation has been commonly used as an effective method to prolong its half-life in serum. In contrast to the nonspecific PEGylation under basic conditions that targets lysine residues randomly, PEGylation sites under mildly acidic conditions preferably targets histidine residues, and there is only one histidine residue at 51 in r-hirudin; therefore, succinimidyl carbonyl methoxy polyethylene glycol (SC-mPEG, 20000) was attached to r-hirudin at mildly acidic pH to favor the formation of mono-PEGylated r-hirudin. The reaction mixture with high mono-PEGylated ratio was easily separated by a one-step ion-exchange chromatographic (IEC) procedure. Approximately 79.71% of the mono-PEGylated r-hirudin was PEGylated at His51, which showed that the acidic PEGylation operation prevented the PEGylation of active center (Lys47) of r-hirudin in principle. Mono-PEGylated product with purity higher than 95% was obtained as the predominant product, and 34% of the anticoagulant activity was retained in vitro. The staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was improved to obtain perfect electrophoretic pattern in less than 5min. More accurate molecular weight was deduced due to the use of PEGs as molecular weight standards.