Abstract: In order to utilize Lactobacillus casei to prepare fermented pineapple juice beverage, the growth curve, acid production, acid tolerance, and sugar tolerance characteristics of Lactobacillus casei LK-1 (L. casei LK-1) were investigated and analyzed in this research. Moreover, the optimal conditions for the fermentation of pineapple juice by L. casei LK-1 were explored by single-factor test and response surface methodology, with the viable cell count and total acid content as indicators. As revealed by the results, L. casei LK-1 grew and reproduced well in an environment with pH5~7 or containing 0% to 10% glucose. The survival rates of it were 76% and 71%, respectively, after 3 h of incubation in an acidic (pH3) or high sugar (40% glucose) environment. Besides, after 48 h incubation in MRS medium, its acid production was 5.35 g/kg, which was good for the fermentation of the juice. The optimal conditions for fermenting pineapple juice with L. casei LK-1 were: Initial pH of 6.8, fermentation temperature of 37 ℃, inoculum level of 1% (v/v), as well as fermentation time of 30 h. Under this preparation condition, the viable cell count of fermented pineapple juice was 8.99±0.04 lg CFU/mL, the total acid content was 7.16±0.26 g/kg, and the juice was bright in color, sweet and sour with excellent flavor. The results of this research can provide a theoretical and technical basis for the further application of L. casei LK-1 in fermented foods.