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Herein, we present the first report on label-free electrochemical sensing of D-Mannitol using MgAl2O4 Nanopowder (MAO) modified glassy carbon electrode (GCE) sensor for pico-molar detection of D-Mannitol in biological samples. The differential pulse voltammetry (DPV) technique is employed for the sensing of D-Mannitol over a wide linear range of detection ranging from 1 pM to 1.5 nM with a sensitivity of 6.47 nA/pM cm−2 and an ultra-low limit of detection (LOD, $3\sigma $ ) of 0.371 pM. The sensor shows an excellent selectivity towards D-Mannitol in the presence of interfering analytes such as glucose, sucrose, human serum albumin (HSA) and bovine serum albumin (BSA). Additionally, the sensor exhibits successful reproducibility with n = 5 devices and excellent stability. This enhanced performance of the MAO/GCE is attributed to the excellent electrocatalytic property of MAO thru Al2+/Al2+ redox couple facilitating the redox reaction of D-Mannitol and the presence of oxy functional groups which improves the adsorption process of D-Mannitol oxidase (mannose) onto the surface of MAO/GCE. The as-fabricated sensor could successfully determine D-Mannitol in the biological samples with excellent recovery percentages from ~98.2% to ~110.42% thus proving to be an excellent platform for developing low cost, high-performance bioanalytical sensing systems.