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The purpose of this study was to analyze the potential of Eleutherine bulbosa (Mill.) Urb. extract as prebiotics for aquaculture organisms by in vitro. The bulbs of E. bulbosa was extracted using 96% ethanol by maceration method. The ability of extract of E. bulbosa as a prebiotic were analyzed the activity to stimulating the growth of Pseudoalteromonas piscicida 1Ub and Bacillus sp. NP5, hydrolysis activity of artificial gastric acid and α-amylase. The results showed the oligosaccharides content included inulin, fructooligosaccharide (FOS), galaktooligosaccharide (GOS) and rafinose of 2.1%, 10%, 1% and 7.5%, respectively. The mixed oligosaccharides were found capable to stimulating the growth of Pseudoalteromonas piscicida 1Ub and Bacillus sp. NP5. The optimum extract of E. bulbosa concentration to stimulate the growth of probiotic was 1.25 mg/mL with score of 0.17 ± 0.02 and 0.21 ± 0.00, respectively. The mixed oligosaccharides were also resistant to hydrolysis by artificial gastrice acid and α-amylase, giving maximum hydrolysis of 8.25 ± 0.25% and 27.52 ± 1.68%, respectively. The results of this study showed that extract of E. bulbosa has potential as prebiotics for aquaculture organisms.