A highly sensitive amperometric L-glutamate biosensor based on the electrocatalytic oxidation of reduced nicotinamide adenine dinucleotide has been developed on Lauth's Violet (known as thionine)/multiwalled carbon nanotubes (Th-MWCNTs) composite film, which is used as a mediator and an enzyme immobilization matrix. The glutamate biosensor, which is fabricated by immobilizing glutamate dehydrogenase (GLDH) on the surface of Th-MWCNTs, displayed a precipitous response (ca. 3 s), a low detection limit (15.9 nM), a wide linear dynamic range (0.1 to 500 mu M), and high sensitivity of 281.6 mu AmM-1 cm(-2), higher biological affinity, as well as good stability and repeatability. Interferences from other biological compounds were also studied for the fabricated sensor. The Th-MWCNTs system exemplifies a simple and efficient approach to the assimilation of GLDH and electrodes, which can provide analytical access to a large group of enzymes for wide range of bioelectrochemical applications in health care fields.