[Background] Recently, the medicinal value of calcified alcohols analogues (active vitamin D and its analogues) in the treatment of tumors therapy and immune regulation has gradually been discovered and examined. However, as the current method for chemical synthesis is complex and difficult, more efficient preparation method of calcified alcohols analogues becomes a hot research focus. [Objective] In order to bioconvert the substrates of calcified alcohols analogues using Pseudonocardia sp. and further improve the bioconversion rate. [Methods] Raw materials and intermediates commonly used in chemical synthesis were bioconverted with Pseudonocardia sp. to find appropriate substrates. The bioconversion conditions were optimized by single factor method and orthogonal test. [Results] Alfacalcidol (1α-(OH)VD3), eldecalcitol intermediate (AD-M07) and paricalcitol intermediate (PC-M07) were bioconveted into calcitriol (1α,25(OH)2VD3), eldecalcitol intermediate (AD-M08) and paricalcitol (PC). The optimal bioconversion conditions of the above three substrates (1α-(OH)VD3, AD-M07, PC-M07) were as follows: peptone 20.0 g/L, glucose 15.0 g/L, PMCD 0.5%, temperature 25 °C to 30 °C, inoculation quantity 5% to 10%, bioconversion time 72, 72, 96 h, substrates concentration 1.2, 0.6, 0.6 mg/ml, initial pH 6.0 to 8.0. Under the above conditions, the maximum bioconversion rate of the three products is 85%, 96%, 75% respectively. [Conclusion] By optimizing the bioconversion conditions, the bioconversion rate of the three products was markedly increased, which provides a good reference for more efficient preparation of calcified alcohols analogues by bioconversion.