A procedure for preparing individualized chitin nanofibers 3-4 nm in cross-sectional width and at least a few microns in length was developed. The key factors to prepare the chitin nanofibers with such high aspect ratios are as follows: (1) squid pen beta-chitin is used as the starting material and (2) ultrasonication of the beta-chitin in water at pH 3-4 and 0.1-0.3% consistency for a few minutes. Transparent and highly viscous dispersions of squid pen beta-chitin nanofibers in water can be obtained by this method. No N-deacetylation occurs on the chitin molecules during the nanofiber conversion procedure. Moreover, the original crystal structure of beta-chitin is maintained, although crystallinity index decreases from 0.51 to 0.37 as a result of the nanofiber conversion. Cationization of the C2 amino groups present on the crystallite surfaces of the squid pen beta-chitin under acid conditions is necessary for preparing the nanofibers.