We report a new fabrication approach to highly sensitive optical sensors by combining the techniques of electrospinning and electrostatic layer-by-layer adsorption. A fluorescent probe, hydrolyzed poly[2-(3-thienyl) ethanol butoxy carbonyl-methyl urethane] (H-PURET), was electrostatically assembled onto the surface of cellulose acetate (CA) electrospun nanofibrous membranes. The fluorescence of these membranes can be quenched by extremely low concentrations (ppb) of methyl viologen (MV2+) and cytochrome c (cyt c) in aqueous solutions. This high sensitivity is attributed to the high surface-area-to-volume ratio of the electrospun membranes and efficient interaction between the fluorescent conjugated polymer and the analytes.