A simple and novel amperometric biosensor for glucose detection is proposed. It is based on the immobilization of glucose oxidase (GOx) in a poly(vinyl alcohol) (PVA) matrix directly drop casted on a platinum electrode surface (Pt/GOx-PVA). Glucose was determined in the absence of a mediator used to transfer electrons between the electrode and the enzyme. The correlation between peak current (i(p)) and scan rate has been verified and the effect of pH solution has been checked. Glucose detection has been performed amperometrically at -400 mV by using pulsed amperometric detection (PAD). Under the selected optimal conditions, the biosensor showed low detection limit (10 μM), wide dynamic range (0.1-37 mM) and high sensitivity. The biosensor amperometric response revealed it to be specific to glucose without significant interference from other sugars and electroactive species coexisting with glucose in biological fluids. Response stability was another interesting feature of the developed system as it was almost completely recovered when the biosensor was left in opportune storage conditions (i.e., a response decrease of only 13% after 35 days in air at room temperature). Finally, X-Ray Photoelectron Spectroscopy (XPS) characterization revealed a homogeneous film deposited on the Pt substrate whose structure is also preserved under operative conditions.