Due to their programmable structures, many aptamers can be readily split into two halves while still retaining their target binding function. While split aptamers are prevalent in the biosensor field, fundamental studies of their binding are still lacking. In this work, we took advantage of the fluorescence enhancement property of a new aptamer named OTC5 that can bind to tetracycline antibiotics to compare various split aptamers with the full-length aptamer. The split aptamers were designed to have different stem lengths. Longer stem length aptamers showed similar dissociation constants (Kd) to the full-length aptamer, while a shorter stem construct showed an 85-fold increase in Kd. Temperature-dependent fluorescence measurements confirmed the lower thermostability of split aptamers. Isothermal titration calorimetry indicated that split aptamer binding can release more heat but have an even larger entropy loss. Finally, a colorimetric biosensor using gold nanoparticles was designed by pre-assembling two thiolated aptamer halves, which can then link gold nanoparticles to give a red-to-blue color change.