Hydantoin hydrolase with responsibility for the ring opening of hydantoin is one of the components of hydantoin utility enzymes of Arthrobacter BT801 which can convert 5-benzylhydantoin into L-phenylalanine. The expression of hydantoin hydrolase gene (hyuH) is very important in elucidation of mechanisms of bio-catalysis and its application in asymmetry synthesis of amino acids. To improve the production and activity of the enzyme, the hydantoin hydrolase gene was amplified by PCR and cloned into E. coli by using vector pT221. The hydantoin hydrolase gene was highly expressed in E. coli BL21 (DE3) under the control of T7 promoter. A protein band about 50kD was detected by SDS-PAGE in the recombinant cell lysate. The objective protein in BL21 (DE3)/pT221-hyuH accounted for 40% of total cellular protein, mostly in soluble form. The products in the recombinant strain showed biological activity.