We had earlier reported the construction and characterization of a high affinity recombinant scFv generated from a potential neutralizing mouse monoclonal antibody against the Hepatitis B surface antigen. In this report we describe the humanization of this scFv by grafting its antigen binding site onto framework of the human consensus sequence of highest similarity. We have used molecular modeling to alter not only the clearly permissible residues but also several minimal positional template and V(H)/V(L) interface residues. The humanized scFv retains the binding characteristic of the mouse monoclonal even under conditions that usually destabilize antigen antibody interactions. This high affinity humanized scFv provides a basis for the development of prophylactic/therapeutic molecules.