Poly[tris((p-aminophenoxy)phosphineoxide)-3,3',4,4'-benzophenonetetracarboxylicdiimide] (PAB) was used as an immobilized enzyme membrane for the fabrication of an amperometric glucose sensor. Enzyme immobilization was performed by two different methods and structural morphology of immobilized enzyme membrane was studied by SEM. The amperometric response of the sensor to hydrogen peroxide, formed as the product of the enzymatic reaction, was measured at a potential of 0.7 V in phosphate buffer solution by means of the TB technique. The influence of preparation conditions (polyimide film thickness, enzyme amount) on electrode performance was examined to obtain the optimal experimental parameters. Sensor characteristics (pH, response time, selectivity, stability) of the PAB/GOx/Pt electrode prepared under optimal conditions has been investigated. Results showed that this sensor exhibited a linear range up to 6 mM and a response time of about 60 s. with good stability. When glucose was injected into the PBS solution in the presence of fouling substances (lactose, sucrose, and urea), excellent amperometric responses were observed. Also, it was seen that the PAB/GOx-Pt electrode blocked the signal current of electroactive oxalic acid.