We report a general approach to light-emitting polymer nanofibers (PNFs) based on waveguiding excitation. By waveguiding excitation light along the PNFs, we demonstrated that the interaction of light with PNFs is enhanced over 3 orders of magnitude compared with the currently used irradiating excitation. Intriguing advantages such as enhanced excitation efficiency, low excitation power operation down to nW levels, tightly confined excitation with low cross talk, and high photostability of the light-emitting PNFs are obtained. The waveguiding excitation allows incorporation of various fluorescent dyes into PNFs to generate multicolor emitting sources covering the entire visible spectrum. The light-emitting single PNFs via waveguiding excitation may find widespread nanophotonic applications in chemical and biological sensors, multicolor emitting sources, and lasers.