Nanomaterials have been studied widely as the supporting materials for enzyme immobilization because in theory, they can provide low diffusion resistance and high surface/volume ratio. Common immobilization methods, such as physical adsorption, covalent binding, crosslinking, and encapsulation, often cause problems in enzyme leaching, D structure change and strong mass transfer resistance. We have previously demonstrated a site-specific enzyme immobilization method, which is based on the specific interaction between a His-tagged enzyme and functionalized single-walled carbon nanotubes (SWCNTs), that can overcome the foresaid constraints. In this work, we broadened the use of this immobilization approach by applying it on other nanomaterials, including multi-walled carbon nanotubes and carbon nanospheres. Both supporting materials were modified with Nα,Nα-bis(carboxymethyl)-l-lysine hydrate prior to enzyme immobilization. The resulting nanomaterial–enzyme conjugates could maintain 78–87% of the native enzyme activity and showed significantly better stability than the free enzyme. When compared with the SWCNT–enzyme conjugate, we found that the size variance among these supporting nanomaterials may affect factors such as surface curvature, surface coverage and particle mobility, which in turn results in differences in the activity and stability among these immobilized biocatalysts.