The development of a low-cost, robust, and versatile biosensors for the rapid detection of endogenous and exogenous metabolites in small animals is of great interest for health-care, pharmaceuticals, and in translational medicine. This work presents a complete in vitro characterization of a system of membranes for the development of a biosensor that will be integrated with the dedicated electronics into an implantable device for small animals. The system of membrane consists of an “inner” permselective layer, designed to filter the signal generated by the oxidation of interfering substances present in biological fluids; an “outer” layer made by an epoxy-enhanced polyurethane film, that regulates the passage of glucose and oxygen to the electrode surface and provides a biocompatible layer for a correct integration with the surrounding tissue. This system of membrane is employed in a glucose sensor that successfully monitors glucose in both the human and mouse physiological range, in PBS and in human serum at 37 ◦C. The sensor showed good stability for 30 days, and the permselective membrane effectively filtered out ascorbic acid and uric acid. Moreover, with the same system of membrane, we developed a biosensor for detection of the anti-inflammatory drug acetaminophen. The integration with the dedicated electronics is successfully used to measure glucose in the physiological range.