Recombinant Anti-Ad fiber knob x Anti-EPCAM Bispecific Antibody (Fab-Fv) is designed to be expressed as two polypeptide chains. The heavy chain Fd fragment and light chain of an anti-Ad fiber knob antibody are fused with the VL and VH domains of an anti-EPCAM antibody respectively. It can form natural in vivo heterodimerization. This BsAb can retarget of adenoviral vectors to tumour cells. It can be used in cancer gene therapy to transfer a therapeutic gene into tumour cells aiming at selective and efficient cell killing.