A Box-Behnken design (BBD) was used for optimization of enzymatic hydrolysis of waste cotton fibers in order to obtain finest cellulose particles. The effect of three factors including hydrolysis time (h), substrate concentration (g/l) and enzyme loading (%) were investigated. The median size of the hydrolyzed particles was defined as the response function of the design. Results of BBD were subjected to analysis of variance (ANOVA) and a quadratic polynomial equation was developed for predicting the particle size. According to the fitted model, the optimal conditions i.e. hydrolysis time, substrate concentration and enzyme loading were suggested as 175 h, 5 g/l and 2.3 %, respectively. The suspension prepared under optimum conditions was subjected to ultrasonic treatment, and the resulted stable suspension of cellulose nanoparticles was characterized. It was found that these nanoparticles are spherical and most of them are in the range of 40-90 nm. The enzymatic and ultrasonic treatments caused an increase in the crystallinity and a decrease in the degree of polymerization of cotton.