An easy covalent immobilization method used to develop enzyme biosensors based on carbon and gold screen printed electrodes (SPCEs and gold SPEs) is described. The linkage of biomolecules through 4-nitrobenzenediazonium tetrafluoroborate, mercaptopropionic acid and thioctic acid monolayers has been attempted using bare SPCEs and gold SPEs, as well as gold nanoparticles (AuNPs) modified SPCEs and gold SPEs. Direct covalent attachment of Cytochrome P450 2B4 (CYP450 2B4) to the transducer has been carried out by carbodiimide and hydroxysuccinimide. Experimental variables in the immobilization process and in the chronoamperometric determination of Phenobarbital (PB) have been optimized by the experimental design methodology. Reproducibility of the different biosensors has been checked under the optimum conditions, yielding values lower than 6%. Their performances have been shown by the determination of PB in pharmaceutical drugs.