Bispecific antibody (BsAb) fragments, such as bispecific tandem scFv and bispecific diabody (Db), have a convenient size that allows for rapid tissue penetration and high target retention. Among BsAb fragments, the bispecific Db and bispecific tandem single-chain Fv (taFv) formats are the most widely used. To prevent homodimer formation, a single-chain Db (scDb) was developed by connecting two hetero-scFv fragments with an additional middle linker. The domain order consisting of BsAb fragments can be varied, and several reports have suggested that this order affects both expression levels and function. However, the order of the most functional domains varies depending on the antibody clones used to construct BsAb fragments and their targets, making it difficult to predict. Both bispecific scDb and taFv formats have eight possible domain orders, which also affect expression levels and function. Here, we demonstrated a method for rearranging the domain order and functionally evaluating bispecific scDb and taFv formats.