Bispecific antibodies (BsAbs), capable of directing T cells to kill specific cancer cells by transiently binding the two cell types, have emerged as one class of promising cancer immunotherapies. However, their wide clinical application might be hampered by two deficiencies: high cost and inconvenience in drug administration. This study presents concept-proving data that these problems could be bypassed by using an enhanced nonviral DNA vector minicircle (MC) to produce BsAb in vivo. It was found that the anti-CD3/CD20 produced from the minicircle (MC.CD20) could effectively mediate the T-cell killing of multiple CD20-positive human B-cell lymphoma cell lines in vitro. More importantly, it was demonstrated that delivery of 5 μg of MC.CD20 to mouse liver via hydrodynamic injection resulted in both the expression of a therapeutic level of anti-CD3/CD20 throughout the 32-day experiment and effective anticancer activity in a B-cell lymphoma xenograft mouse model. The data suggest that MC encoding the BsAbs may become an attractive cancer immunotherapy modality based on its excellent features of safety, efficacy, and convenience in both preparation and use, and its affordability once the delivery technology matures.