We report the preparation of anisotropic chitosan nanofibers prepared using an electrospinning technique. The effect of electrospinning on the formation of nanofibers was examined from results of bulk chitosan. The morphological, structural characterizations and thermal properties of the chitosan bulk and electrospun nanofibers were analyzed by field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetry (TGA), Fourier transform infrared (FTIR), and Raman spectroscopy. Matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) was performed to accurately characterize the high aspect ratio nanofiber structure by the direct identification of mass resolved chains. FE-SEM showed that the electrospun chitosan nanofibers had diameters ranging from 10 to 1,200 nm with an anisotropic nature. MALDI-TOF revealed the presence of lower mass group protonated amino groups, which was the main constituent for the formation of the ultrafine nanofibers in chitosan.