A novel nanostructure of flexible Janus nanofibers with tunable and enhanced magnetism-photoluminescence difunctionality has been successfully fabricated by one-pot electrospinning using specially designed parallel spinneret. Europium complex Eu(BA)(3)phen (BA = benzoic acid, phen = 1,10-phenanthroline), terbium complex Tb(BA)(3)phen, and Fe3O4 nanoparticles (NPs) were, respectively, incorporated into polyvinyl pyrrolidone (PVP) and electrospun into Janus nanofibers. The morphology, structure, and property of the Janus nanofibers were investigated in detail by scanning electron microscopy, energy dispersive spectroscopy, transmission electron microscopy, fluorescence spectroscopy, and vibrating sample magnetometry. The average diameter of the Janus nanofibers is about ca. 300 nm. Every Janus nanofiber is composed of one nanofiber with different performances on two sides, one side is composed of Fe3O4 NPs and PVP, and the other side consists of [Eu(BA)(3)phen + Tb(BA)(3)phen] and PVP. This novel nanostructures can effectively isolate Eu(BA)(3)phen and Tb(BA)(3)phen from Fe3O4 NPs so that the exciting light and emitting light in the [Eu(BA)(3)phen + Tb(BA)(3)phen]/PVP domain will almost not be affected by Fe3O4 NPs. The Janus nanofibers possess higher fluorescent intensity owing to their special nanostructure compared with non-Janus composite nanofiber with the same compositions. Moreover, the Janus nanofibers have multicolored and color-tunable photoluminescence, the emitting color could be tuned in a wide color range of red-yellow-green by adjusting the molar ratio of Eu(BA)(3)phen to Tb(BA)(3)phen under the excitation of 274-nm single-wavelength ultraviolet light, and the magnetism also could be tuned by changing the amounts of Fe3O4 NPs. The new type Janus nanofibers with tunable and enhanced magnetism-photoluminescence bifunctionality have potential applications in the fields of color display, biology cell separation, drug/gene target delivery, and fluorescence immunoassays/imaging. Besides, this novel strategy and construct technique for the Janus nanofibers is of universal significance for the fabrication of other multifunctional Janus nanofibers.