The fermenting liquor from Aspergillus niger D2-26 was purified to obtain a single protein component, and then the characterization of thermostable lactase was studied. The enzyme had an optimum temperature of activity at 70℃ and an optimum pH of 2.5, and had good temperature tolerance from 30℃ to 60℃ and the pH stability at 2.0~9.0. Mn2+ had a significant activation on lactase activity, whereas the enzyme activity was inhibited strongly by Hg2+、Pb2+ and SDS; the Vmax, Km, molecular mass of single subunit protein and glycosylation of Lactase was 0.097 nmol/min, 8.77 mmol/L, 116.978 kD and 11.3%, respectively.