The measure of sorbitol in serum can act as a good indicator in the monitoring of the diabetic complications. To analyze the sorbitol level in serum medium, fluorometric enzymatic assay was performed. To remove the excess sorbitol from the body, proposed binding of sorbitol with caffeine was investigated. Their interaction in serum medium was studied and established by UV-Vis, fluorescence spectrophotometry, and time-correlated single photon counting (TCSPC). The linear calibration of sorbitol (in the range 10-50 mM) was done using UV-Vis spectrophotometry. Time scan experiments furnished the reaction rate of sorbitol assayed solution as well as sorbitol-caffeine complex as 0.021 min −1 and 0.018 min −1 , respectively. A sudden drop was observed in the fluorescence lifetime of reduced nicotinamide adenine dinucleotide (NADH) present in sorbitol assayed solution upon complexation with caffeine, that is, from 1.774 × 10 −09 to 1.23 × 10 −10 Sec, which indicates the hindrance in the formation of NADH and the probable formation of some other species. Isothermal titration calorimetric experiments clearly indicate the number of binding sites (i.e., 3.89, 1.40, and 2.07) that exist between sorbitol and caffeine at the complexation ratio of 1:1.2, 1:1.5, and 1:3. The present method can be helpful in pharmacological and therapeutic studies of sorbitol using caffeine for treating diabetic neuropathy.