Surface refunctionalized fluoropolymer membranes were applied in the design of whole cell and enzyme biosensors based on the Clark-type oxygen sensor. Fluoropolymer membranes (poty(hexafluoropropylene-co-tetrafluoroethylene)(FEP)) were treated using a recently developed procedure that employs a hydrogen/methanol vapor radio frequency glow discharge plasma to introduce hydroxyl functionality into the polymer backbone in a controlled fashion. Hydroxylated materials were aminated by treatment with (γ-aminopropyl)triethoxysilane (APTES). The surface amine groups served as attachment sites for whole cells and enzymes. Initial work measured the permeability and diffusion coefficients for oxygen in hydroxylated, aminated, and base (nonmodified) FEP membranes. Refunctionalized membranes retained the oxygen permeability and diffusion characteristics of the base fluoropolymer. Subsequent experiments investigated the response of biosensors constructed using aminated FEP as the gas-permeable membrane of a Clark-type oxygen sensor. The respiration of NB2a neuroblastoma cells was recorded following cell attachment to the membrane through natural growth processes. In a quiet solution, the response of the oxygen sensor decreased by ~40% in the presence of a monolayer of respiring cells. Sensor response slowly returned to baseline after the cells were exposed to millimolar levels of sodium azide. The response of an enzyme electrode, constructed by linking glucose oxidase and albumin to free amine sites of aminated FEP, is also demonstrated. The calibration curve for glucose was linear over a concentration range between 0.1 and 6.5 mM, and the sensor response reached a steady state within about 60s of exposure to glucose.