Optimization of enzymatic hydrolysis of wool fiber was carried out using a Central Composite Design (CCD) in order to produce wool nanoparticles. The effects of three important determinants, i.e. enzyme loading, substrate concentration and hydrolysis time on enzymatic efficiency were investigated. Polynomial regression model was fitted to the experimental data to generate predicted response such as particle size. The results were subjected to analysis of variance (ANOVA) to determine significant parameters used for optimization. Wool nanoparticles was produced under the attained optimal condition (enzyme loading: 3.3%, substrate concentration: 5 g/l and hydrolysis time: 214 h), followed by ultrasonic treatment. SEM micrographs indicated wool fiber degradation in which the outer cuticle layer was removed and the inner cortical cells were isolated. The results of particle size analysis indicated the positive effect of sonication on reducing particles size further. FTIR spectra denoted no evident changes in the composition of the chemical groups in the macromolecular structure of wool fiber. Besides, the enzymatic hydrolysis and ultrasonic treatment led to an increase in crystallinity, solubility in caustic solution and thermal stability of wool nanoparticles, but caused a decrease in moisture regain comparing to the raw wool fiber. (C) 2012 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.