The purpose of this work is to develop a bioanode using the enzymes of glucose oxidase (GOx) and alcohol dehydrogenase (ADH) as catalysts to oxidised glucose and alcohol present in different beverages. The study was carried out using the covalent bonding method for both enzymes via the functionalization of carbon nanofibers for the formation of carboxyl groups that can form bonds with the amine groups of the enzyme, as well as using tetrabutylammonium bromide (TBAB) with Nafion. The optimum operation parameters of both enzymes (pH and temperature) were determined for the later evaluation in a microfluidic fuel cell. In addition, using the scanning electrochemical microscopy technique, a local study of enzymatic processes is used to demonstrate that the enzymes immobilized on the same electrode remain active. The evaluation of the microfluidic fuel cell was carried out using different solutions, 0.01 M glucose, 0.01 M ethanol and a mixture of 0.01 M glucose and 0.01 M ethanol, all in phosphate buffer solutions at pH 7, where it was possible to obtain a maximum performance of 5.07 ± 0.1 mW cm−2, and there was a significant increase in current density compared to non-composite solutions (glucose or ethanol). In addition, different alcoholic beverages were used to evaluate the versatility and adaptability of the bi-enzymatic anode electrode with the perspective use in Lab-on-a-Chip systems.