Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA af?nity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 °C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 °C and 6.7, respectively. The Km of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 μmol/(min·mg). Besides, α-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of α-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6?6.5 and 25 °C for 21 h. Under these conditions, α-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of α-arbutin was about 31 g/L.