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In this report, we present an enzyme deposited Au electrode for an electrochemical measurement of acetylacetic acid (AcAc) in urine. The electrode has an immobilized layer of a mixture of D-β-hydroxybutyrate dehydrogenase (HBDH) and nicotinamide adenine dinucleotide (NADH) as sensing material to investigate its electroanalytical properties by means of cyclic voltammetry (CV). The modified electrodes are used for the detection of AcAc and present a linear current increase when the AcAc concentration increases. The electrode presents a limit of detection (LOD) of 6.25 mg/dL in the range of 6.25–100 mg/dL for investigation of clinical relevance. Finally, the electrode was evaluated using 20 patient samples. The measured results of urine ketone by the developed electrode were compared with the clinical results from a commercial kit, and the analysis showed good agreement. The proposed electrode was demonstrated to be a very promising platform as a miniaturized electrochemical analyzer for point-of-care monitoring of the critical biochemical parameters such as urine ketone.