This paper describes the development and optimization of an amperometric bienzymatic biosensor for glucose, based on glucose oxidase (GOX) and peroxidase (HRP), immobilized on mesoporous silica with hexagonal symmetry (MCM‐41). The mesoporous material was immobilized by Nafion assisted adsorption on a glassy carbon electrode, which was used as working electrode. Measurements were performed in a conventional three electrode cell using a Ag/AgCl reference electrode and platinum wire as counter electrode. Operating parameters such as GOX/HRP enzyme ratio, mediator (catechol) concentration, applied potential and pH were optimized. The proposed biosensor showed excellent analytical performance with detection and quantification limits of 8.6×10−6 M and 2.5×10−5 M, respectively; analytical sensitivity of 4.67×10−4 AM−1, repeatability <5 % RSD and a long‐term stability of 10 days (5 daily measurements). Also, five possible interferences were studied and only ascorbic acid showed an amperometric signal, which can be removed with 5 % iodine pretreatment. The biosensor was successfully tested for glucose determination in pharmaceutical formulations, and compared well with an established commercial photometric enzymatic kit (Megazyme).