In this study, we demonstrated a novel fabrication method of three dimensional nanoporous gold thin film (NPGF) onto gold (Au) substrate using electrochemical deposition method. Scanning electron microscope (SEM) investigation reveals the formation of highly-ordered pores, approximately 30 nm in diameter and 150 nm thick. The NPGF-modified electrode shows a linear range (0.1–40 μM) for dopamine detection in the presence of ascorbic acid. The electrochemical measurements of mixtures of dopamine, ascorbic acid, and uric acid in human serum sample for real sample applications was also investigated based on differential pulse voltammetry (DPV) technique. These high sensitivity and selectivity features of the proposed NPGF biosensor offer great promise for real sample biosensor application.