There is an urgent need to develop low cost electrochemical sensors wherein the sensor can be disposed after recording data, thereby eliminating the issue of inaccuracy arising from repeated sensing measurements, which plagues most conventional electrochemical sensors. This work is the first demonstration of a NiSe2 based disposable, one time use electrochemical glucose sensor in bio-mimicking real samples wherein NiSe2 was hydrothermally grown NiSe2 on a biodegradable cellulose paper. Both physicochemical (x-ray diffraction, x-ray photoelectron spectroscopy, field emission scanning electron microscope) and electrochemical (impedance spectroscopy and cyclic voltammetry (CV)) characterization techniques confirmed the growth and presence of NiSe2 on a cellulose paper. Electrochemical techniques like CV and amperometric (i-t) were utilized for the selective and sensitive oxidation of glucose. The results suggests that the proposed NiSe2 sensor is effective in a linear range of 0.1-1 mM with fast response time (3.9 s), low detection limit (24.8 ± 0.1 μM) and high sensitivity (0.25 A M-1 cm-2) at a potential applied (E app = 0.55 V versus Ag|AgCl). Prior to the real sample analyses i.e. glucose detection in human urine, the fabricated NiSe2 sensor was tested for selectivity towards glucose in co-existing interferences (dopamine, ascorbic acid, uric acid, urea, sodium chloride, fructose, lactose and cysteine). Finally, glucose in artificial blood serum and urine samples was demonstrated with the fabricated NiSe2 sensor and the results are comparable to the conventional laboratory methods. The present methodology presents a novel possibility towards the design of next generation, affordable point-of-care devices for a broad range of clinical diagnostics. © 2019 IOP Publishing Ltd.