Take the yield of 3β,7α,15α-trihydroxy-5-androsten-17-one( 7α,15α-diOH-DHEA) as index,suitable methods and materials for the immobilization of Colletotrichum lini ST-1( C. lini ST-1) were selected. Then,the impact of immobilization conditions,conversion conditions and reused batches on immobilized cells were investigated. The results showed that,cells entrapted in sodium alginate( SA) were proved to be more appropriate,and the optimized immobilization conditions were SA 3%( m /v) and CaCl25%. The optimum transformation conditions of dehydroepiandrosterone( DHEA) by immobilized cells were as follows: temperature 27 ℃,shaker speed 180 r /min,buffer concentration 20 mmol /L,12 g of immobilized beads /20 mL buffer solution,addition of 11 mmol /L glycerol,9% FeSO4and 2% PEG400. On above condition,when 8 g /L DHEA was added,the yield of the product were up to71. 2%,increased 47. 5% compared with using free cells. Batch conversion results showed that the best concentration of substrate was 10 g /L. At the same time,the immobilized cells could be reused for 4 batches. The capacity of the immobilized cells was 1. 09( g product /g cells),which was 2 times higher than that of free cells.