Since cellulose nanofiber (CNF) has unique characteristics in terms of renewability, high specific elastic modulus and strength and transparency, it is attractive for a building block of future materials. CNF can be extracted from various natural resource by several means. However, the size of the extracted CNF is very broad and uniformity of the extracted CNF is very important for many applications. Thus, a fractionation process is necessary to obtain a uniformly sized CNF. In this paper, a simple centrifugal fractionation was carried out to reduce the size distribution of the extracted CNF suspension from hardwood pulp by the combination of TEMPO oxidation and aqueous counter collision methods. The original CNF suspension was diluted and centrifuged under low speed to remove cellulose microfibers then centrifuged under high speed to separate very small CNF. The centrifugation condition is 10 k rpm for 1 h followed by 45 k rpm for 4 h. The fractionated CNF was analyzed by an atomic force microscopy, and the length and width distribution histogram analysis was utilized. UV-visible analysis, FT-IR and XRD crystallinity analysis were carried out to analyze all fractionated CNFs and the original CNF. After centrifugal fractionation, the width and length distribution range were reduced by 62% and 70%, respectively. It is shown that the centrifugal fractionation is an easy and efficient method to fractionate a uniform CNF suspension.