A new fluorescent glucose assay has been created using Alexa Fluor 647-labeled concanavalin A (Con A) and a fourth-generation PAMAM Alexa Fluor 594-labeled glycodendrimer. This assay has been shown to have a large response to glucose within the biological range and to be capable of functioning within a polymer hydrogel. In this paper, the glucose response is shown to be a single fluorophore-based quenching reaction. Data showing that the sensor is fully reversible and specific through competitive binding between the dendrimer and glucose with Con A are presented. Overall, the assay is shown to have potential over the traditional dextran-based assay because it has a larger dynamic response to physiological glucose concentrations, incorporates longer wavelength dyes that improve signal penetration through dermal tissue, and provides an internal reference in the form of a nonreactive fluorescent label.