[Objective] Cloning of a new gene possessing a function of Echinocandin B (ECB) side-chain deacylation from a wild type Streptomyces strain NCPC-1020 of soil source. [Methods] Using degenerate PCR and TAIL PCR strategy, we successfully cloned the target gene, which was heterologously expressed in S. lividans TK24, then a whole-cell catalysis method was developed to dectect the deacylation activity of ECB deacylase by LC-MS. [Results] The ECB deacylase gene was cloned and its function was confirmed. [Conclusion] Combined the two PCR techniques, degenerate and TAIL PCR, a new gene could be cloned quickly. The obtain of the new ECB deacylase gene lays a good foundation for the researches on semibiosynthetic of ECB related drugs.