Targeting to two or more objectives simultaneously has been pursued as a strategy to potentially increase the efficiency and selectivity of targeted drugs to certain cancers. In this study, an epidermal growth factor receptor (EGFR)/CD13-targeting, bispecific fusion protein ER(Fv)-LDP-NGR consisting of an anti-EGFR single-chain variable fragment (scFv), an apoprotein (LDP) of lidamycin (LDM), and a tri-CNGRC (Cys-Asn-Gly-Arg-Cys) peptide against CD13 was constructed, and then an enediyne-energized analog ER(Fv)-LDP-NGR-AE was generated by integration with an enediyne chomophore (AE) derived from LDM. The apoprotein LDP was used as a 'scaffold' to connect the scFv fragment and the tri-CNGRC peptide and also served as a specific 'carrier' for the extremely potent cytotoxic enediyne chromophore of LDM. Compared with its monospecific counterparts, ER(Fv)-LDP and LDP-NGR, the bispecific fusion protein ER(Fv)-LDP-NGR showed higher affinity to EGFR/CD13-overexpressed tumor cells. Determined by the MTT assay, the bispecific, enediyne-energized ER(Fv)-LDP-NGR-AE showed highly potent cytotoxicity to EGFR/CD13-overexpressed MCF-7 cells, with an IC50 value of 3.4×10 mol/l, whereas for the EGFR-overexpressed A431 cells, the IC50 value was 2.2×10 mol/l. For MCF-7 cells, the bispecific ER(Fv)-LDP-NGR-AE was more potent in cytotoxicity than the corresponding monospecific energized fusion proteins. In athymic mice models, the bispecific fusion protein ER(Fv)-LDP-NGR presented stronger inhibitory activity than the monospecific ER(Fv)-LDP and LDP-NGR. For the enediyne-energized fusion proteins, ER(Fv)-LDP-NGR-AE significantly inhibited the growth of EGFR/CD13-overexpressed MCF-7 xenograft and EGFR-overexpressed A431 xenograft by 86.3 and 81.4%, respectively. In addition, the bispecific ER(Fv)-LDP-NGR-AE showed much higher efficacy than its monospecific analogs ER(Fv)-LDP-AE and LDP-NGR-AE in both MCF-7 and A431 xenograft models. The results show that EGFR/CD13 bitargeting effectively improved the antitumor efficacy. Both the bispecific fusion protein and its enediyne-energized analog are highly effective in athymic mice bearing xenografts, and the latter exerts more marked efficacy. Generation of a pair of bispecific antibody-based therapeutics and its corresponding antibody-drug conjugate simultaneously may be a feasible strategy for the development of new targeted drugs for cancer therapy.