Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (VH) and variable light (VL) domains tethered together by a flexible peptide linker. We have characterized the individual VH or VL domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both VH and VL domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the VH or the VL domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active VH or VL domains can occur by partnering with different VH or VL complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors.