Lignin plays a crucial role in enzymatic hydrolysis of lignocellulosic biomass. To evaluate the correlation between lignin properties and its effects on enzymatic hydrolysis, five organosolv lignins (OLs) were isolated from woody biomass, and their physico-chemical properties and structural features were characterized. The effects of OL addition on enzymatic hydrolysis of microcrystalline cellulose (pure cellulose) were assessed first, which showed their disparate effects. The addition of three OLs increased the 72 h hydrolysis yield by 7.4% to 10.1%, while the addition of other two OLs reduced the 72 h hydrolysis yield by 3.2% to 20.4%. A strong correlation between the enzyme distribution coefficient on lignins and the 72 h hydrolysis yields indicated that the enzyme-lignin interaction played a significant role in determining the lignin effects. More importantly, a correlation between lignin properties (hydrophobicity, zeta potential, and particle size) and the enzyme distribution coefficient was established. Identifying the key lignin properties will give insights to reduce the lignin inhibition by altering the lignin properties, thereby promoting enzymatic hydrolysis of lignocellulose.