This work aimed to investigate the effects of various single factors and their interactions on the preparation of cellulose nanofiber (CNF) using ball milling. These factors included the milling ball size, milling time, mass ratio of milling ball to cellulose pulp, and alkali concentration, as well as their complicated interactions. The Taguchi method was applied to the experimental design to systematically study the influences of these factors on the yield of CNF. A method for evaluating the yield of CNF was developed. Both of the simple analysis (SA) and analysis of variance (ANOVA) of the experimental results indicated that the most significant factor influencing the yield of CNF was the mass ratio of milling ball to cellulose pulp. The interactions between this factor and other single factors, e.g., milling time, also showed significant influence on the yield of CNF. Although the influence of milling ball size was negligible, its interaction with mass ratio of milling ball to cellulose pulp must be considered during production. Based on these findings, a mini scaled-up practice was proposed to demonstrate the possibility of the application of ball milling on the mass production of CNF.