This work demonstrates the single-step solvothermal synthesis of marcasite FeS<sub>2</sub> as a low-cost electrochemical platform for the separation-less sensing of four nucleobases of DNA. FESEM micrographs reveal high surface area micro-flowers like morphology of the marcasite FeS<sub>2</sub> wherein each micro-flower comprises vertically grown interwoven 2D nanosheets. XRD and Raman spectra further confirm the successful synthesis of the marcasite FeS<sub>2</sub>. A detailed optimization study on the impact of electrolytic pH values on the simultaneous determination of four DNA bases (adenine (A), thymine (T), guanine (G) and cytosine (C)) is performed. Under optimal pH conditions, the marcasite FeS<sub>2</sub> based electrode is capable of identifying DNA bases altogether over the linear range of 500-2000 µM with excellent reproducibility and stability. The superior electrochemical sensing properties of the marcasite FeS<sub>2</sub> towards simultaneous detection of four bases can be ascribed to high electro-active surface area and conductivity from the active sites of this unique 2D nanosheets-like structure. Besides, to establish its efficacy in real-time applications, the sensor is successfully utilized for the detection of nucleobases of denatured DNA of calf-thymus, and the obtained value of <inline-formula><tex-math notation="LaTeX">$\frac{{( {G + C} )}}{{( {A + T} )}}$</tex-math></inline-formula> is found to be 0.77. These results establish that the marcasite FeS<sub>2</sub> has enormous potential to be used as a promising low-cost, functional transition metal di-chalcogenide (TMD) nanomaterial for bio-analytical and sensing applications.