The availability of self-targeting and low immunogenic therapeutic agents is critical to efficient cancer therapy. Therefore, the development of humanized therapeutic antibodies is particularly appealing. A humanized single-chain variable fragment (scFv) antibody that can target human epidermal growth factor receptor-2 (HER2)-overexpressing cancer cells was designed and produced via expression in Pichia pastoris. The expression gave a high yield of 8 mg protein/l (with a purity of 92 %) using shake-flask cultures. Functional studies also revealed that the purified recombinant anti-HER2 scFv exhibited anti-proliferative activity and could bind efficiently to HER2-overexpressing human breast cancer cell line SKBR3. The recombinant scFv offers promising therapeutic and binding efficiencies that are desirable for targeted cancer therapies.