Novel flexible Janus nanofibers with magnetism-upconversion luminescence bifunction have been successfully fabricated via electrospinning technology using a homemade parallel spinneret. NaYF4:Yb3+, Er3+ and Fe3O4 nanoparticles (NPs) were, respectively, incorporated into polyvinyl pyrrolidone (PVP) and electrospun into Janus nanofibers with NaYF4: Yb3+, Er3+/PVP as one strand nanofiber of the Janus nanofiber and Fe3O4/PVP as the other one. The morphologies and properties of the final products were investigated in detail by X-ray diffractometry, scanning electron microscopy, transmission electron microscopy, energy dispersive spectrometry, vibrating sample magnetometry and fluorescence spectroscopy. The results reveal that Janus nanofibers simultaneously possess superior magnetic and upconversion luminescent properties due to their special nanostructure, and the upconversion luminescent characteristics and saturation magnetizations of the Janus nanofibers can be tuned by adjusting the content of NaYF4 : Yb3+, Er3+ NPs and Fe3O4 NPs. Compared with Fe3O4/NaYF4 : Yb3+, Er3+ /PVP composite nanofibers, the magnetism-upconversion luminescence bifunctional Janus nanofibers provide better performances owing to isolating NaYF4 : Yb3+, Er3+ NPs from Fe3O4 NPs. The novel magnetism-upconversion luminescence bifunctional Janus nanofibers have potential applications in the fields of new nano-bio-label materials, drug target delivery materials and future nanodevices owing to their excellent magnetic-upconversion luminescent properties, flexibility and peculiar nanostructure. More importantly, the new strategy and construction technology are of universal significance to fabricate other bifunctional Janus nanofibers.