We report the construction, expression and purification of a bispecific single-chain Fv antibody fragment produced in Escherichia coli. The protein possesses a dual specificity: the single-chain FvB1 portion is directed to the Idiotype of BCL1 lymphoma cells, the single-chain Fv2C11 moiety binds to the CD3 marker on T cells. The two domains are joined by a flexible peptide linker. Using Immobilized Metal Affinity Chromatography, the recombinant protein was purified from bacterial insoluble membrane fractions. After refolding of the bispecific protein, it was affinity-purified. As demonstrated by flow cytometry, both binding sites are retained in the refolded protein. Retargeted cytotoxicity and T cell proliferation assays further prove the biological activity and specificity of the bispecific single-chain Fv. Thus, these bispecific molecules show a potential anti-tumor activity.