The release of insulin from the pancreas is tightly controlled by glucokinase (GCK) activity that couples β-cell metabolism to changes in blood sugar. Despite having only a single glucose-binding site, GCK displays positive glucose cooperativity. Ex vivo structural studies have identified several potential protein conformations with varying levels of enzymatic activity, yet it is unclear how living cells regulate GCK cooperativity. To better understand the cellular regulation of GCK activation, we developed a homotransfer Förster resonance energy transfer (FRET) GCK biosensor and used polarization microscopy to eliminate fluorescence crosstalk from FRET quantification and improve the signal-to-noise ratio. This approach enhanced sensor contrast compared to that seen with the heterotransfer FRET GCK reporter and allowed observation of individual GCK states using an automated method to analyze FRET data at the pixel level. Mutations known to activate and inhibit GCK activity produced distinct anisotropy distributions, suggesting that at least two conformational states exist in living cells. A high glucose level activated the biosensor in a manner consistent with GCK's enzymology. Interestingly, glucose-free conditions did not affect GCK biosensor FRET, indicating that there is a single low-activity state, which is counter to proposed structural models of GCK cooperativity. Under low-glucose conditions, application of chemical NO donors efficiently shifted GCK to the more active conformation. Notably, GCK activation by mutation, a high glucose level, a pharmacological GCK activator, or S-nitrosylation all shared the same FRET distribution. These data suggest a simplified model for GCK activation in living cells, where post-translational modification of GCK by S-nitrosylation facilitates a single conformational transition that enhances GCK enzymatic activity.