Gliomas are rarely curable malignant brain tumors arising from normal glial cells. The scorpion-derived small peptide, chlorotoxin (CTX), can selectively bind malignant gliomas. In the present study, a CTX-conjugated onconase (Onc), a small cytotoxic ribonuclease, was prepared as a potential anti-glioma drug. In this conjugate, recombinant CTX was covalently linked with recombinant Onc by reversible disulfide linkage. The chemically conjugated CTX-Onc showed much higher cytotoxicity to the cultured glioma U251 and SHG-44 cells than the physical mixture of CTX and Onc (CTX + Onc). In the nude mouse models bearing subcutaneous U251 or SHG-44 tumors, the CTX-Onc conjugate also showed improved anti-tumor effects than the CTX + Onc control. These results suggested that the reversible chemical-conjugated CTX promoted the tumor targeting of Onc, and thus the present CTX-Onc conjugate could be further developed as a potential targeted anti-glioma drug.