The binding interaction of N-Phenylbenzohydroxamic acid (PBHA) with Ct-DNA was measured by four methods, (i) UV absorption, (ii) fluorescence emission, (iii) viscosity and (iv) DNA melting technique. The molecular docking of PBHA with the DNA was performed using 3D-FFT mode and the differences in their binding modes were investigated The result revealed intercalations exist in the system. The values of binding constant, Kb, is 2.01x104 l M-1 and Stern Volmer constant Ksv is 1.41 x104 l M-1 obtained by UV absorption and fluorescence spectral methods, respectively. The thermodynamic parameters ∆H, ∆S, ∆G were calculated, which suggested that hydrogen bond play major role in the binding of DNA with PBHA. The binding interaction trend is further confirmed by viscosity and DNA melting technique performed on PBHA–Ct-DNA complex which shows the increase in viscosity and DNA melting as a function of PBHA concentration.