In this study, the immobilization technology was used to improve the LA yield and shorten the fermentation time. The optimum conditions to immobilize Lactobacillus pentosus ATCC 8041 cell were determined by Taguchi design L16 (4). The immobilized L. pentosus ATCC 8041 cells prepared by 2% sodium alginate (SA) and 6% polyvinyl alcohol (PVA) with the immobilization process by 0.10 M calcium chloride (CaCl) and 2.5% boric acid (HBO) had the best performance of LA yield at the temperature of 35 °C, which is significantly higher than that of L. pentosus ATCC 8041 free cells. These cells maintained the stable and efficient performance in 15 repeated batch fermentation, and they also have excellent mechanical strength to keep from breakage caused by cell growth and agitation.