Abstract <p>Onconase (ONC), a novel antitumor protein, exhibits significant cytotoxic effects on various tumor cells. Although recent advancements have been made in ONC-based molecularly targeted drugs, most are single-targeting proteins with limitations such as poor targeting precision and low efficiency. In this study, the fusion protein rONC-T7-pHLIP was constructed by linking ONC with the T7 peptide, which specifically recognizes the transferrin receptor, and the pHLIP peptide, which actively targets the acidic tumor microenvironment, using a flexible linker peptide (GGGGS)₃. The engineered strain <i>E. coli</i> BL21(DE3) / rONC-T7-pHLIP was used for single-factor analysis of IPTG concentration, induction time, and induction temperature, followed by orthogonal experimental design to optimize the expression conditions, resulting in a 10% increase in fusion protein expression. Cytotoxicity and flow cytometry apoptosis assays demonstrated that the purified fusion proteins—dual-targeting rONC-T7-pHLIP and single-targeting rONC-T7 and rONC-pHLIP—exhibited significantly higher antitumor activity against cancer cells compared to native ONC, with the dual-targeting variant showing superior efficacy over the single-targeting ones. Immunofluorescence assays confirmed that rONC-T7-pHLIP binds to cancer cells and exerts its activity in the cytoplasm. In conclusion, these findings suggest that the novel fusion protein rONC-T7-pHLIP has potential as a targeted therapeutic agent for cancer treatment.</p> Key points <p>• <i>The rONC-T7-pHLIP was expressed in the E. coli expression system with high yield.</i></p> <p>• <i>The rONC-T7-pHLIP showed high stability and safety in vitro.</i></p> <p>• <i>The rONC-T7-pHLIP significantly improved the antitumor activity of cancer cells.</i></p>