The fish disease epidemiology is urgent to be investigated for the surveillance and prevention. The diseased fish showed splenomegaly with diffusion of white nodules and microscopical granulomatous formation. It is important to develop a method of pathogens isolated from clinical samples with serial dilution method and disc diffusion method. Representative colonies were selected from diseased cobia on BHIA plate and were inoculated onto MacConkey agar, TCBS agar, and blood agar. The cage-culture of the different bacterial groups detected in the survey of bacteria isolated from THOD, HDSB, EMD with serial dilution method. 119 from 128 isolated strains were Gram¡¦s negative (93%), including pathogenic Vibrio spp. 57% (73/128) in THOD. 54 from 90 isolated strains were Gram¡¦s negative (60%), including pathogenic Vibrio spp. 12.2% (11/90) in HDSB. 61 from 104 isolated strains were Gram¡¦s negative (59%), including pathogenic Vibrio spp. 70.2% (77/90) in EMD. In different times diseased grouper, 104 from 139 isolated strains were Gram¡¦s negative (75%), including pathogenic Vibrio spp. 88% (123/139), in 2003. While 24 from 44 isolated strains were Gram¡¦s negative (55%), including pathogenic Vibrio spp. 73% (32/44), in 2004. 66 from 75 isolated strains were Gram¡¦s negative (88%), including pathogenic Vibrio spp. 97% (73/75) by disc dilution method in EMD. 9 from 31 isolated strains were Gram¡¦s negative (30%), including pathogenic Vibrio spp. 26% (8/31), by disc dilution method of grouper in PCG. A DGGE (denaturing gradient gel electrophoresis) technique can identify six groups of bacteria from cobia, and J6, R13, T29 have similarity 100%. Quantity One Version 4.5 (Bio-Rad) can identify six groups of bacteria from diffusion methods that F group diluted the bacterial strain from serial dilution method. B group and E group diluted the bacterial strain from disc diffusion method. Higher resistance rates of the different bacterial strains isolated from cobia and were £]-lactam and susceptible were observed in quinolones.