Purpose: Magnetic nanofibers (MNFs) are nanofibers impregnated with magnetic nanoparticles (MNPs). Magnetic particle imaging (MPI) is a recently introduced imaging method that allows imaging of the spatial distribution of MNPs. The purpose of this study was to develop MNFs and to investigate the feasibility of visualizing them using MPI and of heating them using an alternating magnetic field (AMF). Materials and Methods: First, chitosan nanofibers were cross-linked with glutaraldehyde vapor in a sealed vial for 24 hours. Next, they were mixed with various concentrations of MNPs and the mixture was stirred for 1 hour using a magnetic stirrer. After the mixture was refrigerated at -80藲C for 24 hours, it was freeze-dried for 24 hours. The morphology of the resultant MNFs was characterized by scanning electron microscopy, and the magnetic properties were measured using a vibrating sample magnetometer. After these measurements, we imaged the MNFs using our MPI scanner, and investigated the correlation between the pixel values of the MPI image and the concentration of MNPs or the number of MNF sheets. We also heated the MNFs using AMF, and measured the temperature rise using an infrared thermometer. Results: The MNFs were successfully visualized using our MPI scanner, and the pixel values of the MPI image showed excellent correlation with the concentration of MNPs (r = 0.992) and the number of MNF sheets (r = 0.997). A significant temperature rise was observed under AMF, and the initial slope of the time-dependent temperature rise showed excellent correlation with the concentration of MNPs (r = 0.994) and the number of MNF sheets (r = 0.979). Conclusion: The MNFs developed in this study can be visualized using MPI and can be applied to magnetic hyperthermia. They will be useful in biomedicine including cancer therapy and tissue regeneration.