Electrochemical biosensors (an integrated system of biological constituent and physiochemical detector) have attracted plenty of attention due to its high precision, and importance in clinical diagnosis. In electrochemical biosensors, redox indicators like methylene (MB) blue are used to detect the electrical changes, which by intercalating between DNA base pairs, can produce an intense redox signal on the surface of ssDNA modified electrode. This study reports the interaction between calf thymus DNA (ctDNA) and new methylene blue (NMB), having almost identical basic skeleton to MB. Various techniques like UV-Visible, thermal melting, fluorescence, circular dichroism (CD) spectroscopy and molecular docking have been utilized. Hypochromism and a red shift in UV-Vis spectra revealed the intercalation binding mode for DNA-NMB complex. Nearly identical binding constants were calculated using UV-Visible and fluorescence spectroscopy. The calculated thermodynamic parameters like change in enthalpy (ΔH°) and entropy (ΔS°) were found to be -6.11×104 and -128.96 JK-1mol-1 at 290 K respectively. CD revealed a change into more compact B-DNA conformation after binding with NMB. These studies suggest that intercalation mode, hydrogen bonding, and van der Waals forces might be responsible for DNA-NMB interaction. This work might further facilitate our understanding about DNA-drug interactions for utilizing them for medicinal purposes and nanochemistry.