Gelatinases play important roles in tumor progression and are abundantly expressed in a variety of malignant tumors. Antibody targeting gelatinases is a possible avenue to fight against cancer. However, antibody alone can not achieve curative efficacy. Herein, we demonstrated the intensified targeting therapy of a tandem scFv-based fusion protein and its enediyne-energized analogue against gelatinases-overexpressed tumor. A fusion protein dFv-LDP, comprising a tandem scFv of anti-gelatinases linked to the apoprotein (LDP) of lidamycin, was generated and showed strong tumor targeting capability in three different tumor xenografts. In PG-BE1 lung carcinoma xenograft, the tumor inhibition rate was 77.5% by dFv-LDP versus 94.2% by dFv-LDP-AE, the product of dFv-LDP assembled with the active enediyne chromophore (AE) of lidamycin. Moreover, the combination of dFv-LDP with dFv-LDP-AE further augmented the therapeutic efficacy, producing initial tumor shrinkage in five of six mice. The microvessel density (P