Electrospun Ru(bpy)₃²⁺-doped Nafion (ERDN) nanofibers were successfully fabricated by a one-step electrospinning technique. The diameters of the nanofibers range from 120 nm to 220 nm. The ERDN nanofibers, evenly distributed on the substrate with their random orientations, form a porous 3-D structure nanofibrous membrane. Compared to continuous thin films, the obtained nanofibrous membrane maintains a higher surface-area-to-volume ratio, a larger amount of immobilized-Ru(bpy)₃²⁺, and a better stability of the Ru(bpy)₃²⁺ immobilization. As a result, the nanofibers' ECL signal is enhanced ∼15-fold and stable (does not change during continuous CV scans for 50 cycles). As a sensing platform, the nanofibers can sensitively detect low concentration phenolic compounds by monitoring the phenol-dependent ECL intensity change. The detection limit for phenol is 1.0 nM based on a signal/noise ratio >3, which is comparable or better than that in reported phenol assays. In addition, the nanofibers exhibit excellent ECL behaviors on Au, Pt, GC and ITO electrodes. A great potential for ERDN nanofibers-based ECL sensors is offered.