Abstract: Yeast β-glucan was a multifunctional polysaccharide, but it was insoluble in water and present particles. To broaden its applicability, the yeast β-glucan nanoparticles were prepared by ball milling pulverization using yeast cells of Saccharomyces cerevisiae, and their physicochemical properties were studied. Taking the average particle size as an index, the effects of some factors such as deproteinization, freezing and thawing on pulverization of yeast β-glucan particles were studied, and the ball milling process was optimized. The results showed that long-term high-temperature extraction, deproteinization, repeated freezing-thawing and chlorine dioxide treatment were beneficial to pulverizing of yeast β-glucan particles, which made the particle size of yeast β-glucan was significantly reduced after ball milling. The optimum ball milling conditions of yeast β-glucan were rotating speed 850 r/min, ball-to-material ratio 10:1, and ball milling time 120 min. Under the above suitable conditions, the average particle size of freeze-dried yeast β-glucan nanoparticles was 81.16±0.35 nm, and its purity was 89.52%. The infrared spectrum analysis showed that yeast β-glucan nanoparticles still maintained its original basic chemical structure. Yeast β-glucan nanoparticles had perfect suspension stability in aqueous solution. Compared with yeast β-glucan, yeast β-glucan nanoparticles had higher oil-holding capacity, but lower water-holding capacity and viscosity. Yeast β-glucan nanoparticles were successfully prepared by ball milling pulverization, and their physicochemical properties changed obviously. The results of this study provided a basis for further investigating and developing yeast β-glucan.