Measurement of glucose is an important component of care in the intensive care unit. Current methodologies for glucose monitoring include enzymatic-based laboratory analyzers, point of care testing and continuous glucose monitoring systems that all require breaking the skin. This study focuses on the development of a glucose biosensor to measure transdermal glucose (TG) by noninvasive passive diffusion. The H152C glucose binding protein (GBP) labeled with the polarity-sensitive dye BADAN (6-bromoacetyl-2dimethylaminonaphthalene) was prepared and characterized with the aim of producing a biosensor with an operating range at micromolar levels compatible with TG concentrations. The BADAN-labeled GBP exhibited a large fluorescence intensity increase (228%) upon addition of a saturating concentration of glucose. The glucose binding constant was calculated to be (K d ) 1.124 ± 0.2361 M. The biosensor has a linear operating range of 0.030-0.460 M, making it suitable for monitoring transdermal glucose. The use of the biosensor was demonstrated by measuring TG collected by passive diffusion of glucose through the skin of healthy adult subjects. Results showed that the H152C GBP labeled with BADAN is capable of measuring TG and can be used for noninvasive glucose sensing.