Aim: The objective of this study was to explore isolation, identification, enzymatic and antibacterial activity of intestinal bacteria of Zebra fish (Danio rerio). Methods and Results: The serially diluted (10-6) sample was plated in sterilized nutrient agar medium and incubated at 37oC for 24 hrs. After incubation the pure colonies were characterized for their morphological and various biochemical characteristics using the Berge’s Manual of Determinative Bacteriology. The isolated and identified bacteria was Aeromonas sp., Pseudomonas sp., Micrococcus sp., Escherichia sp., and Bacillus sp., The digestive enzyme productivity of isolated bacteria were identified from the culture plate using selective media. The isolated bacteria were subjected for its efficacy to produce enzymes like amylase, cellulase, lipase and protease. The antibacterial activity of isolated bacteria was tested with five fish pathogenic bacteria (Klebsiella pneumonia, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus and Escherichia coli). Based on the results the selected bacteria was Pseudomonas sp., produced higher enzyme productivity of amylase, cellulose and lipase and also higher zone of inhibition in antibacterial activity test. Conclusion: Based on the above characteristics the selected intestinal bacteria was multiplied and recommended to the preparation of probiotic feed. Significance and Impact of Study: Zebra fish have near by 75% of genome is same in human genome. In future isolated Zebra fish intestinal bacteria has been formulated as probiotic feed preparation and it can be further characteristic and also will produce large scale for enhance the growth.