Carbon nanotubes (CNTs) have been widely studied and used for the construction of electrochemical biosensors owing to their small size, cylindrical shape, large surface-to-volume ratio, high conductivity and good biocompatibility. In electrochemical biosensors, CNTs serve a dual purpose: they act as immobilization support for biomolecules as well as provide the necessary electrical conductivity for electrochemical transduction. The ability of a recognition molecule to detect the analyte is highly dependent on the type of immobilization used for the attachment of the biomolecule to the CNT surface, a process also known as biofunctionalization. A variety of biofunctionalization methods have been studied and reported including physical adsorption, covalent cross-linking, polymer encapsulation etc. Each method carries its own advantages and limitations. In this review we provide a comprehensive review of non-covalent functionalization of carbon nanotubes with a variety of biomolecules for the development of electrochemical biosensors. This method of immobilization is increasingly being used in bioelectrode development using enzymes for biosensor and biofuel cell applications.