Here we report the thermodynamic analysis of glucose interaction on conformational transitions on DNA duplex with different base compositions and lengths by using Circular Dichroism, UV thermal melting and native gel electrophoresis. CD results revealed that three duplexes Duplex 1 “D1” and Duplex 2 “D2” folded into B-form while Duplex 3 “D3” folded into mixed A and B like forms in the presence of different cations and glucose. We monitor the interaction between DNA duplex and glucose over a range of temperatures. Thermal melting data revealed that these duplexes are more stable without glucose under all cationic (Na+, K+, Na+ + Mg++ and K+ + Mg++) conditions tested and calculated the enthalpy “∆H”, entropy “∆S” and free energy “∆G”. Moreover, these duplexes are marginally destabilized at high concentration of glucose. In this study, glucose was considered as an essential model for understanding interactions between interfacial water molecules at hydrophilic surfaces of DNA duplexes.