The electrocatalytic properties of the redox couple Ni(OH)(2)/NiOOH at physiological pH was investigated in the presence of cationic polymeric films providing alkaline microenvironments, with the objective of developing a biosensor for cholesterol and glucose. The Ni(OH)(2)/NiOOH modified electrodes in presence of the cationic polymer overlayers were well characterized by optical and electrochemical techniques. The modified electrodes showed excellent catalytic properties toward hydrogen peroxide oxidation and exhibited a wide linear range compared to the conventional and expensive Pt based catalysts used in H2O2 based sensors. The superb electrocatalysis of hydrogen peroxide could be combined with the enzymatic oxidation of cholesterol and glucose showing, the feasibility of monitoring biological metabolites like glucose and cholesterol using the basic catalyst Ni(OH)(2)/NiOOH at physiological pH. Modified electrodes were highly stable and yielded reproducible results. Further the influence of graphene layer in decreasing the overpotential of the detection of H2O2 and its effect on the biosensing of cholesterol and glucose was further investigated. The sensing protocol was validated in the analysis of blood sugar in serum samples. (C) 2017 The Electrochemical Society.