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A fluorescence based glucose biosensor, for diabetes management, was developed based on a fluorescent resonant energy transfer between fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate. Upon implantation under the skin of a rat, large losses of fluorescent signal resulted in poor signal-to-noise and difficulty in correlating peak ratio to glucose concentration. In an attempt to remedy this issue the use of longer wavelength dyes was implemented in vitro. Experiments were performed to verify the effectiveness of the sensor using AlexaFluor 594 labeled dextran and AlexaFluor 647 labeled Concanavalin A while encapsulated in a poly( ethylene glycol) hydrogel sphere. Using porcine skin tissue, in vitro experiments were performed to quantify the improvement in signal-to-noise and peak resolution. The results indicated that the use of longer wavelength fluorophores allowed for better tissue penetration and in turn increased the overall signal-to-noise of the detection system.