Much recent research has focused on electrochemical biosensors to meet the growing demands for rapid and accurate diagnostic tests for diseases, detecting toxic molecules present in the environment, and biomolecules used as in pharmaceuticals. Carbon nanotubes (CNTs) have been shown to be effective for electrochemical biosensors because of their favorable chemical, physical and electrical properties. Here we report the use of a two-dimensional entangled mat of CNTs, known as buckypaper, as electrodes for electrochemical biosensors. The buckypaper electrodes exhibit superior sensitivity towards tryptophan, L-carnitine, tyrosine and myoglobin. When engineered with metals or other specific molecules, the buckypaper shows an up to 1000-fold increase in the signal compared with electrodes based on glassy carbon. This enhanced electrochemical performance of the modified buckypaper demonstrates potential for a new platform for electrochemical biosensor technologies.