An electrochemical biosensor was developed for the monitoring of the concentration of ketone 3-β-hydroxybutyrate (3HB) in a physiological fluid for the potential diabetic patient management. Current electrochemical detection of 3HB involved at least two stepwise reactions, which may also require a mediator to facilitate the electron transfer. The detection method in this study involved only a single reaction step without any mediator. This biosensor operated at a relatively low electrochemical potential (+200 mV versus Ag/AgCl), and enzyme 3-hydroxybutyrate dehydrogenase (3HBDH, EC 184.108.40.206) was immobilized on thick film screen-printed iridium-modified working electrode detecting NADH (nicotinamide adenine dinucleotide, reduced form), which was the reaction product of 3HB and NAD+ (nicotinamide adenine dinucleotide, oxidized form) in the presence of 3HBDH. Electrochemical measurements showed that this biosensor responded well to 3HB in both phosphate buffer and 100% bovine serum. The reproducibility and the interference of this biosensor were studied and assessed. Spectrometric measurements of the ketone 3-β-hydroxybutyrate were carried out and were used to compare the electrochemical outputs of this biosensor, and the biosensor performed very well compared to the spectrometric study.