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Background: Ascorbic acid (AA), dopamine (DA), uric acid (UA), and nitrite (NO2-) are essential biomarkers for human metabolism and can be used to indicate some chronic diseases and metabolic disorders, including scurvy, Parkinson's disease, hyperuricemia, and kidney disease., Objective: A multifunctional electrochemical sensor that can integrate the detection of these species was constructed using nanoporous gold (NPG) as a recognition element to modify glassy carbon electrode (GCE)., Methods: The electrochemical performance of the multifunctional electrochemical sensor was investigated toward AA, DA, UA, and NO2- in citrate buffer solution (CBS, 100 mM, pH 4.0) and human serum using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods., Results: In the quaternary mixture detection, the resulting NPG/GCE electrode displayed four independent oxidation peaks with wide peak separations. Further, the NPG/GCE electrode showed good linear responses with the sensitivities of 32, 1103, 71, and 147 μA/mM/cm2 and the detection limits of 1.58, 0.17, 0.37, and 0.36 μM for AA, DA UA, and NO2-, respectively. Additionally, the NPG/GCE electrode exhibited great anti-interference and was successfully applied in human serum samples., Conclusions: These results indicate that the NPG/GCE electrode can simultaneously and selectively detect AA, DA, UA, and NO2-, which has the potential for application and diagnosis in the screening and diagnosis of chronic diseases and metabolic disorders., Highlights: A multianalyte electrochemical sensor was fabricated for human metabolites detection. The sensor displayed good performance in the simultaneous detection of AA, DA, UA, and NO2- and applied to human serum samples., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)