Layered double hydroxides (LDH) belong to the class of two-dimensional materials having a wide variety of applications ranging from energy storage to catalysis. Often, these materials when used for nonenzymatic electrochemical glucose sensing tend to be interfering with oxygen evolution reaction (OER), resulting in overestimation of the glucose. Herein, to address this, NiFe-based LDH were selected because of their ability to vary the metal ratios. The synthesized LDH have been characterized using various spectroscopic and microscopic techniques. Among the LDH synthesized, Ni4Fe-LDH have been able to differentiate the glucose oxidation potential and the onset potential of OER with minimum interference. The Ni4Fe-LDH sensor shows a sensitivity of 20.43 μA mM–1 cm–2 in the linear range of 0–4 mM concentrations. To further enhance the sensitivity, composites of reduced graphene oxide (rGO) have been synthesized in situ, and the Ni4Fe/rGO5 composites have shown an increased sensitivity of 176.8 μA mM–1 cm–2 attributed to the charge-transfer interactions. To understand the experimental observations, detailed computational studies have been carried out to study the effect of the electronic structure on the metal ratios of the LDH and its role in differentiating glucose sensing and the oxygen evolution reaction. Along with this, theoretical calculations are also carried out on LDH–graphene composites to study the charge-transfer interactions.