TUDCA was prepared from TCDCA in vitro using the double-enzyme-coupled system.
7伪- and 7尾-HSDH, was fixed by separate immobilization and co-immobilization.
Coenzyme NADP<sup>+sup> regeneration was achieved by the double-enzyme-coupled system.
The double-enzyme-coupled system is promissing for the high efficient preparation of TUDCA in vitro.