Bispecific antibody (BsAb) fragments, such as bispecific tandem single-chain Fv and bispecific diabodies, have a convenient size for rapid tissue penetration and high target retention. Their smaller size also allows them to be expressed in cost-effective microorganisms. However, the production of BsAb fragments using Escherichia coli is often hampered by their expression as insoluble aggregates. In addition, BsAb fragments generally require the fusion of affinity peptide tags, which might negatively impact molecular folding and function and increase immunogenicity, as most of these tags are derived from non-human sequences. The production of BsAb fragments from their Fc fusion with a protease recognition site inserted around the hinge region is an ideal strategy. Protein A purification can be applied to Fc fusion proteins, and BsAb fragments can be prepared by protease digestion. Papain is also applicable for preparing BsAb fragments from Fc fusion; however, undesired digestion due to low sequence specificity is sometimes a problem. Herein, a method using a human rhinovirus 3C protease recognition site is presented as an example of the production of BsAb fragments from Fc fusion.