Hyperbranched polysaccharide from Pleurotus tuber-regium (PTR-HBPS) is a β-glucan with high degree of branching (DB, 0.69) and a molecular weight (Mw) of 31.2 × 105 g/mol with mixed β-1, 4/β-1, 4, 6/β-1, 6 glucosidic linkages. PTR-HBPS was depolymerized by cellulase and β-glucosidase under optimized conditions to form PC (PTR-HBPS depolymerized by cellulase) and PG (PTR-HBPS depolymerized by β-glucosidase) fractions with a minimum Mw of 2.74 × 105 and 3.98 × 105 g/mol, respectively. PC fractions had no significant changes for its primary structure in terms of glycosidic linkages, DB, and triple helical structure, while the DB of PG fractions was reduced to 0.63 with the loss of triple helical structure. Nanoparticles fabricated by PC fractions with zein showed better stability under different pH conditions. Enzymatic depolymerized low Mw β-glucan derived from PTR-HBPS with similar structural characteristics as the native one has potential as nanocarriers for food bioactive substances. •Cellulase could depolymerize Mw of PTR-HBPS up to one-eleventh of its original size.•Cellulase cleaved β-1,4 glucosidic bonds in backbone but did not change configuration.•β-Glucosidase could degrade Mw of PTR-HBPS up to one-eighth of its original size.•β-Glucosidase cuts more side branches from non-reducing end caused a decrease of DB.•PC3/zein nanoparticles have better pH stability.