Glucokinase (GK), an enzyme critical to glucose metabolism, exhibits thermal instability, which can affect its enzymatic activity under physiological and pathological conditions. This study aims to mathematically model the thermal denaturation kinetics of GK and empirically validate the model using experimental data. To establish a mathematical model on thermal denaturation of glucokinase (E.C.2.7.1.2) and its experimental validation, the enzyme glucokinase was investigated in a 0.075M Tris HCl buffer with pH 9.0 at 30∘C and 0.6M MgCl2. A first-order kinetic model was developed to describe the enzyme’s denaturation, incorporating temperature-dependent reaction rates based on the Arrehenius equation. Empirical data were collected through Spectrophotometer across a temperature range of 20∘–60∘C. Experimental validation revealed that GK undergoes irreversible denaturation above 60∘ with a significant reduction as temperature increases. Moreover, the thermal denaturation of GK in the presence of osmolyte Urea is a critical process affecting enzyme stability and function. This study also aims to mathematically model and empirically validate the impact of Urea on GK’s thermal denaturation behavior. Results demonstrated that Urea significantly reduces the thermal stability of GK, lowering its denaturation temperature. The results are simulated graphically using the Wolfram MATHEMATICA software. The mathematical predictions closely matched experimental data, confirming the model’s accuracy.