Herein, a novel electrochemical biosensor has been proposed for highly sensitive and simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA) based on 4-sulfocalix[6]arene-functionalized carbon nanotubes (CNTs/SCX6) decorated with AuPt bimetallic nanoparticles. Ultraviolet-visible (UV-Vis) spectrophotometry, transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS) mapping, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) have been applied to characterize the morphology and structure of CNTs/SCX6-AuPt. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and amperometric i-t curve reveal that CNTs/SCX6-AuPt-modified glassy carbon electrode (GCE) possesses excellent electrooxidation activity toward AA, DA, and UA. The obtained detection ranges of CNTs/SCX6-AuPt for simultaneous detection of AA, DA, and UA are 0.1-2 mM, 1.5-20 mu M, and 2-40 mu M with corresponding detection limits of 5.4 mu M, 0.08 mu M, and 0.37 mu M, respectively. The high electrocatalytic activity and rapid electronic transfer ability of CNTs/SCX6-AuPt result in high selectivity, stability, reproducibility, and repeatability. The constructed electrochemical sensor has been used to simultaneously determine AA, DA, and UA in urine samples with good recoveries, which may become a promising method for trace analysis of biological molecules in the complex physiological environment.