Epidermal growth factor receptor (EGFR), overexpressed in many epithelial tumors, plays important roles in the formation and the development of tumors, and thus it is regarded as a promising target for cancer therapy. Single-chain variable fragment (scFv), an engineered antibody fragment, is generally used for constructing antibody-targeted drugs, owing to its low immunogenicity and high penetration capability into solid tumors. A fusion protein ER(Fv-LDP), consisting of an anti-EGFR scFv and the apoprotein (LDP) of lidamycin (LDM), was prepared and then assembled with the active chomophore [active enediyne (AE)] of LDM to generate enediyne-energized analogue ER(Fv-LDP-AE). The fusion protein ER(Fv-LDP) bound specifically to EGFR-overexpressing cancer cells and internalized into the cytoplasm through receptor-mediated endocytosis. ER(Fv-LDP) possessed cytotoxicity against carcinoma cell lines, which was hundreds of times more potent than the separate moiety of ER(Fv) and LDP. The enediyne-energized fusion protein ER(Fv-LDP-AE) also showed stronger cytotoxicity to target-relevant cancer cells than LDM in vitro. In human epidermoid carcinoma A431 xenografts, ER(Fv-LDP) presented higher antitumor efficacy than that of ER(Fv), LDP, and their mixture, with tumor growth inhibition rates of 63.6, 46.7, 48.5, and 49.9%, respectively. The enediyne-energized fusion protein ER(Fv-LDP-AE) at a dose of 0.4 mg/kg inhibited tumor growth by 89.2%, while no significant body weight loss was seen in treated animals. The results show that an anti-EGFR scFv-based fusion protein and its enediyne-energized analogue can be prepared by DNA recombination and molecular reconstitution. Both ER(Fv-LDP) and ER(Fv-LDP-AE) are effective against EGFR-overexpressing cancer xenograft in athymic mice. An integrated technical platform for scFv-based enediyne-energized fusion proteins has been established.