Glucose is the primary currency of energy in many physiological and cellular processes. Consequently, monitoring glucose is crucial in medical applications and bioprocesses. Over the past few decades, periplasmic binding proteins such as glucose binding proteins (GBP) produced by the E. coli fermentation process have gained significant attention as an alternative to enzymes for glucose measurement. GBP are bacterial receptors that can be fluorescently labeled and undergo conformational changes when they bind to glucose substrates. The glucose concentration can then be quantified by monitoring the fluorescence intensity produced due to the biorecognition event. It has previously been shown GBP as the biorecognition element, can measure glucose with high sensitivity. Here we present a prototype for automatic glucose monitoring based on GBP. Our prototype integrates an aseptic sampling technique to automatically and continuously monitor glucose in a bioprocess. The monitoring setup includes a novel sampling system, a specially modified chromatography column to hold the immobilized GBP, and a micro fluorometer for efficient fluorescence measurement, all of which are combined into a compact portable system. The system establishes a calibration with standard glucose solutions and is utilized to monitor the glucose concentration in yeast culture in a mini bioreactor. The YSI Model 2700 Select Chemistry Analyzer is used to validate the prototype system. For the glucose range of 35 mM to 50 mM, an accuracy within 10 mM was obtained. Moreover, for lower concentrations (20 mM to 35 mM), accuracy improved to within 5 mM of glucose. These results show that our system has the potential to measure glucose accurately at lower concentrations. Additionally, the system is capable of utilizing branched-chain-amino-acid (BCAA) binding proteins to monitor concentrations of leucine, isoleucine, and valine. In the future, this technology can easily be adapted to the measurement of additional analytes such as glutamine, ATP, etc., and for healthcare applications such as non-invasive transdermal glucose monitoring.