In vivo continuous glucose monitoring has posed a significant challenge to glucose sensor development due to the lack of reliable techniques that are non- or at least minimally-invasive. In this proof-of-concept study, we demonstrated the development of a new glucose sensor protein, AcGFP1–GBPcys–mCherry, and an optical sensor assembly, capable of generating quantifiable FRET (fluorescence resonance energy transfer) signals for glucose monitoring. Our experimental data showed that the engineered glucose sensor protein can generate measurable FRET signals in response to glucose concentrations varying from 25 to 800 μM. The sensor developed based on this protein had a shelf-life of up to 3 weeks. The sensor response was devoid of interference from compounds like galactose, fructose, lactose, mannose, and mannitol when tested at physiologically significant concentrations of these compounds. This new glucose sensor protein can potentially be used to develop implantable glucose sensors for continuous glucose monitoring.