A novel single-layered enzyme-membrane using BPPO film which was pore-filled with cross-linked PVA containing TYR was prepared to develop a pesticide biosensor with an increased stability. The prepared enzyme-membrane was assembled on a glassy carbon electrode to detect pesticides. The voltammetric measurements revealed a coupled reaction, an enzymatic oxidation and an electrochemical reduction of catechol transported through the prepared membrane. A detection range of parathion and carbaryl was 0.01-1 ppb and 0.01-10 ppb, respectively. The activity in the enzyme-membrane was maintained for 1 month due to favorable aqueous environment of PVA for enzyme activity while BPPO film provided structural stability. (C) 2009 Elsevier B.V. All rights reserved.