A facile approach was developed for the preparation of nanocomposite based on β-lactoglobulin (BLG)-functionalized multi-wall carbon nanotubes (MWCNTs) and gold nanoparticles (GNPs) for the first time. Owing to the amphipathic nature, BLG can be adopted onto the surface of MWCNTs to form BLG-MWCNTs with uniform dispersion in water. Taking advantage of sulfhydryl groups on BLG-MWCNTs, GNPs were decorated on the BLG-MWCNTs-modified glassy carbon electrode (GCE) by electrodeposition. The nanocomposite was characterized by transmission electron microscopy, scanning electron microscopy and X-ray spectroscopy analysis. Cyclic voltammetry and chronoamperometric method were used to evaluate the electrocatalytic ability of the nanocomposite. Furthermore, a glucose biosensor was developed based on the immobilization of glucose oxidase with cross-linking in the matrix of bovine serum albumin (BSA) on the nanocomposite modified GCE. The resulting biosensor exhibited high sensitivity (3.98 μA mM(-1)), wider linear range (0.025-5.5 mM), low detection limit (1.1 μM at the signal-to-noise ratio of 3) and fast response time (within 7s) for glucose detection.