Abstract: OBJECTIVE To achieve tumor microenvironment-sensitive drug delivery which was prepared by recombinant and PEGylation techniques. METHODS A substrate peptide sequence of asparaginyl endopeptidase (legumain) was introduced to the C-terminus of trichosanthin(TCS) by recombinant technique; the recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. TCS-Asn10-Cys recombinant protein was expressed and purified according a standard protocol. Further, the C-terminus thiol of the recombinant protein was conjugated to mPEG-Hz-Mal. The TCS-Asn10-Hz-PEG conjugate was purified using CM sepharose fast flow column. The pH-sensitivity and enzyme-sensitivity of TCS-Asn10-Hz-PEG in vitro were tested. RESULTS The TCS-Asn10-Cys fusion protein was successfully expressed and purified. The tumor microenvironment-drug delivery system of TCS-Asn10-Hz-PEG was prepared. In either the pH 5.6 medium or the presence of legumain, the PEG chain could be cleaved by hydrolysis or proteolysis from the TCS, of which the activity was recovered. CONCLUSION It is developed a dual-sensitive drug delivery system of TCS-Asn10-Hz-PEG that can be responsive to either acidic hydrolysis or legumain proteolysis.