Abstract Amperometric biosensors for the determination of phenolic compounds (phenol, o-cresol, p-cresol, m-cresol, catechol, dopamine, and ephinephrine) have been constructed. It comprises a multiwalled carbon nanotube (MWNTs) conduit, a Nafion backbone, and a tyrosinase function. The modification of glassy carbon electrode is made by a simple solution-evaporation method. The resulting multiwalled carbon nanotube-Nafion-tyrosinase (MWNT-Nafion-Tyr) nanobiocomposite film modified electrode is inexpensive, reliable, and easy to use. The homogeneity of the MWNT-Nafion-Tyr nanobiocomposite films was characterized by atomic force microscopy (AFM). The measurement of phenolic compounds is based on the signal produced by the electrochemical reduction of o-quinones, the product of the enzymatic reaction. Optimization studies were performed in stirred solution and the magnitude of response currents were found to be dependent on applied potential and enzyme loading. The optimized biosensor for phenol determination displayed a sensitivity of 303 μA mM −1 and a response time of less than 8 s.