α-Hydroxynitrilelyase (HNL), a vital tool in the bio-synthesis of optically active cyanohydrin, which can catalyze carbonyls and HCN selective forming optically active cyanohydrin. HNL gene was amplified by PCR using pET30a-HNL as template and then ligated into pMD18-T vector. Validated HNL gene by sequenced was linked into pHMOXGαA vector. Positive recombinants grown in the medium containing 4 mg/mL of G418 were screened using PCR. Bio-active α-hydroxynitrilelyase expressed in Hansenula polymorpha after 0.5% methanol induction was obtained with 2.015 U determined by analysis of enzyme activity and SDS-PAGE.