Single-chain antibodies (scFv) consist of the light and heavy variable domains of antigen binding IgG antibodies tethered by a short peptide linker. The length and composition of this linker can affect the properties of the scFv, thus we have proposed that a scFv can be used as a peptide linker conformational change sensor. A previous study reported the use of an elastin-like peptide linker to confer thermal responsiveness to scFv antigen binding, but did not analyze the role of linker length on this effect. We examine the relative importance of peptide linker length and composition for scFv based conformational change sensor performance. The anti-fluorescein scFv 4D5Flu is used because bound fluorescein has a significantly lower quantum yield compared to free fluorescein, thus binding and unbinding events can be observed by measuring the change in fluorescence. This work represents a proof of principle that 4D5Flu can be used as a conformational change sensor for thermally responsive peptides located in its linker region. At 25^◦C, fluorescein binding affinities for all flexible, constrained, and elastin-like peptide linker mutants were similar (K_d = 20.5-54.7 nM). When the linker length becomes prohibitively short, inactive antibodies form active dimers, consistent with solution based binding affinities. However, when the temperature is raised, antibodies with elastin-like peptide linkers release more fluorescein than mutants with flexible or constrained linkers of equal length for all mutants studied. The most responsive mutant (an elastin 10mer) exhibited a 28% increase in fluorescein release compared to the flexible 10mer. This increase provides a means of separating temperature responsive peptides from random peptide libraries. Dynamic light scattering experiments elucidate the mechanism for this thermally responsive behavior. Insight into the mechanism of thermal responsiveness should provide information crucial to the design of an appropriate selection method for the directed evolution of stimulus and/or biomolecule responsive peptides. These peptides could find application in targeted drug delivery systems, nanodevice actuators, and ‘smart' hydrogels.