The in vitro analytical performance of fluorescence-based oxygen sensing polymeric films prepared with silicone rubbers that spontaneously release nitric oxide (NO) is examined. The use of NO-release polymers for fabricating functional optical sensors is proposed as a potential solution to fingering biocompatibility and concomitant performance problems encountered with prototype intravascular optical oxygen sensors. Plasticized silicone rubber films formulated with two distinct types of diazeniumdiolate NO donors release NO for more than 24 h. The optical oxygen sensing films prepared by doping these NO release polymeric materials with oxygen indicators (pyrene/perylene donor/acceptor pair) display different analytical responses, as compared to controls without NO release capability. Nonlinear Stern-Volmer behavior is observed for single-layer NO release oxygen sensors owing to heterogeneous environments for the pyrene/perylene pair and a concomitant quenching of the fluorescence by excess amine sites in such films. In contrast, a dual-layer configuration using an underlying NO-release silicone rubber layer covered with a second polymeric layer containing the fluorescent indicators is shown to yield identical sensitivity and linearity toward oxygen as conventional non-NO-releasing oxygen sensing films, while still providing surface NO fluxes necessary to yield more thromboresistive devices.