In the present work, the interaction of phosphorous heterocycle (PH) with calf thymus DNA (CTDNA) has been studied using spectroscopy and verified by molecular modeling which is found to be in consonance with each other. Apparent association constant (K = 4.77 × 10 M), calculated using UV-Vis spectra indicating an adequate complex formation between CTDNA and PH. A dynamic mode of the fluorescence quenching mechanism in case of ethidium bromide (EB) + CTDNA by PH has been observed confirming formation of DNA-PH complex. A moderate binding constants of PH with CTDNA + EB has been observed (2.74 × 10 M at 293 K) by means of fluorescence data. Calculated values of thermodynamic parameters enthalpy change (ΔH) and entropy change (ΔS), suggests weak (van der Walls like) force and hydrogen bonds playing the main role in the binding of PH to CTDNA. Furthermore, the results of circular dichroism (CD) reveal that PH does not disturb native conformation of CTDNA. As observed from absorption and fluorescence spectroscopy the binding mode of PH with DNA was indicative of a non-intercalative binding, which was supposed to be a groove binding. The molecular modeling results show that PH is capable of binding DNA having docking binding energy = -7.26 kcal × mol. Above mentioned experimental results are found to be in consonance with molecular docking simulations and supports the CTDNA-PH binding. Graphical Abstract.