Lactic acid bacteria are used popularly for fruit juice fermentation because they are good sources of probiotics for human. In this study, ten lactic acid bacteria were isolated from 7 samples of naturally fermented pineapple juice. All isolates could grow in the MRS medium pH 1.5 and reach high densities (6.80 – 6.95 log CFU/mL) after 2 h of incubation at 37°C. Moreover, Lactobacillus sp. Y1 produced highest lactic acid concentration (1.20% w/v) and identified as L. acidophilus. The different diluted ratios of pineapple juice (0, 10, 20 and 30% w/v) and different sucrose supplementations (0, 3, 6, and 9% w/v) were used for testing fermenting capacity of L. acidophilus Y1. The undiluted pineapple juice with 9% (w/v) of sucrose supplementation was found to be suitable for fermentation. Based on the results of sensory evaluation and bacterial density determination, the favorable conditions for pineapple fermentation were determined as follow: initial bacterial level at 5.0 log cells/mL, fermentation time for 36 h at 37°C. The results of storage testing showed that the suitable temperature for product storage was 4 – 6°C, bacterial density (8.06 log CFU/mL) of final product was maintained up to 3 weeks.