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Phenylketonuria (PKU) is characterized by an autosomal recessive mutation in the phenylalanine hydroxylase (PAH) gene. Impaired PAH enzyme activity leads to the accumulation of phenylalanine (Phe) and its metabolites in the bloodstream, which disrupts the central nervous system and causes psychomotor retardation. Early diagnosis of PKU is essential for timely intervention. Moreover, continuous monitoring of blood Phe levels is indispensable for prognosis, requiring a robust and reliable monitoring system. This study presents an automated lab-on-a-CD-based system for early diagnosis and monitoring of PKU treatment. This miniaturised system contains CD-shaped disposable cartridges, a mini centrifuge, and an electrochemical sensing unit. Modified screen-printed gold electrodes were used for the electrochemical measurements in cartridges. Electrode modification was conducted by electrochemical graphene oxide reduction and deposition on the electrode surface, which increased the sensitivity of the measurement 1.5 fold. The system used amperometric detection to measure Phe in the blood through oxidation of NAD + to NADH by the enzyme phenylalanine dehydrogenase. The limit of detection (LOD), limit of quantification (LOQ), and sensitivity of the system were 0.0524, 0.1587 mg dL -1 and 0.3338 μA mg -1 dL, respectively, within the 0-20 mg dL -1 measurement range ( R 2 = 0.9955). The performance of the lab-on-a-CD system was compared to the gold standard HPLC method. The accuracy was 83.1% for HPLC and 84.1% for the lab-on-a-CD system. In conclusion, this study successfully developed a portable diagnostic device for rapid (under 20 min), accurate and highly sensitive detection of Phe in whole blood.