A new amperometric biosensor for glucose was developed based on adsorption of glucose oxidase (GOx) at the gold and platinum nanoparticles-modified carbon nanotube (CNT) electrode. CNTs were covalently immobilized on gold electrode via carbodiimide chemistry by forming amide linkages between carboxylic acid groups on the CNTs and amine residues of cysteamine self-assembled monolayer (SAM). The fabricated GOx/Au(nano)/Pt(nano)/CNT electrode was covered with a thin layer of Nafion to avoid the loss of GOx in determination and to improve the anti-interferent ability. The immobilization of CNTs on the gold electrode was characterized by quartz crystal microbalance technique. The morphologies of the CNT/gold and Pt(nano)/CNT/gold electrodes have been investigated by scanning electron microscopy (SEM), and the electrochemical performance of the gold, CNT/gold, Pt(nano)/gold and Pt(nano)/CNT/gold electrodes has also been studied by amperometric method. In addition, effects of electrodeposition time of Pt nanoparticles, pH value, applied potential and electroactive interferents on the amperometric response of the sensor were discussed. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for glucose in the absence of a mediator. The linear range was from 0.5 to 17.5mM with correction coefficient of 0.996. The biosensor had good reproducibility and stability for the determination of glucose.