A novel glucose biosensor was constructed, based on the immobilization of glucose oxidase (GOx) with cross-linking in the matrix of biopolymer chitosan (CS) on a glassy carbon electrode (GCE), which was modified with gold-platinum alloy nanoparticles (Au-PtNPs) by electrodeposition on multiwall carbon nanotubes (CNTs) in CS film (CNTs/CS). The properties of Au-PtNPs/CNTs/CS were characterized by scan electron microscopy (SEM), X-ray diffraction (XRD), cyclic voltammetry (CV), and electrochemical impedance spectra (EIS). Primary study indicated that Au-PtNPs/CNTs had a better synergistic electrocatalytic effect on the reduction of hydrogen peroxide than did AuNPs/CNTs or PtNPs/CNTs at a low applied potential window. With GOx as a model enzyme, a new glucose biosensor was fabricated. The biosensor exhibited excellent performances for glucose at a low applied potential (0.1V) with a high sensitivity (8.53 microA mM(-1)), a low detection limit (0.2 microM), a wide linear range (0.001-7.0 mM), a fast response time (<5s), and good reproducibility, stability, and selectivity. In addition, the biosensor was applied in the determination of glucose in human blood and urine samples, and satisfied results were obtained.