The genomic DNA sequence of an organism specifies the features of its vender, but knowing this string of letters by no means tells you the exact Mother Nature of the conforming organism. This present study deals with to Isolation, characterization and molecular identification of Microorganisms from Intestine of Gut from fish by using 16DNA Sequencing. Bacterial strain was isolated and characterized using various biochemical tests and confirmed through molecular approach. Bacterial 16S r DNA gene was amplified using suitable primers. The amplified 16S r DNA gene sequence was compared with the sequence in NCBI sequence database and Phylogenetic and molecular evolutionary analyses were conducted using 16S r DNA sequencing. There are different organisms were isolated and identified the three of its bacillus spand remaining one is Pseudomonas sp. Bacillus sp. is a gram positive, oval shaped bacteria which forms white raised irregular colony. The Pseudomonas is a gram negative aerobic gammaproteo bacteria. The sequence when submitted to NCBI gene bank database using BLAST showed 99 – 100% maximum identityand query coverage.