A characterization method for a C-terminal region of recombinant protein is studied. Recombinant protein was specifically digested by protease to peptide fragments, and then the target C-terminal peptide was purified with affinity chromatography by anti-peptide antibody column from the peptide fragments and analyzed by HPLC. From the retention time and the peak height, the correctness and amount of the C-terminal peptide were determined. With combining the N-terminal sequence method, the soundness and amount of recombinant proteins can easily be determined.