A new amphiphilic lactobionamide functionalized by a pyrrole group has been synthesized and electrochemically characterized. This functionalization has allowed the preparation of poly(pyrrole-lactobionamide) films by oxidative electropolymerization in aqueous and organic electrolytes. The electrochemical immobilization of glucose oxidase (GOx) and polyphenol oxidase (PPO) is carried out in polymeric ＇bilayer＇ and ＇trilayer＇ matrices based on poly(pyrrole-ammonium) and poly(pyrrole-lactobionamide) films. The amperometric response of GOx-based biosensors to glucose in phosphate buffer was based on the oxidation at + 0.6 V vs. SCE of the generated H2O2. A marked enhancement of the biosensor sensitivity (+ 60%) was observed for a trilayer matrix illustrating the beneficial effect of the polymerized lactobionamide groups. The amperometric response of PPO-based biosensors to catechol in chloroform was based on the reduction at - 0.25 V vs. SCE of the generated o-quinone. In the same way, the presence of poly(pyrrole-lactobionamide) as a polymeric additive induces a gnat enhancement of the biosensor sensitivity (+ 350%) in anhydrous chloroform.