The fourfinger threadfin (Eleutheronema tetradactylum) is one of the most important marine fish species, that contributes great economic values to Taiwanese coastal fisheries. Over-feeding results in the shorten growing period, however that induces the high stress in digestion tract of fish, and causes the intestinal diseases to occur frequently. The objectives of this study were to evaluate the effects of probiotic in application with the commercial feed in fourfinger tetradactylum. Three probiotic strains, Bacillus amyloliquefaciens, Lactobacillus Paracasei and Leuconostoc mesenteroides B4, were selected and evaluated on growth rate, survival rate, feed efficiency and feed conversion. The results of growth rates, feed conversion rates and survival rates showed that there was no statistically significant difference between the feed probiotic supplementation groups and the control group. The survival rates of the probiotic feed with Ba group was 48% after being challenge with the high dose Streptococcus iniae which was significantly higher than the 24% of the control group (p<0.05). The low-dose challenge control group has higher organ isolation rates than the higher-dose control group, which shows that the low-concentration streptococcus has a prolonged disease course, leading to the growth of pathogenic bacteria in the fish organs.The results of intestinal lesions showed that the lesions of the intestinal mucosal epithelium of the probiotic feed supplementation groups were all milder than those of the control group, indicating that the test groups still have considerable protective ability in the intestines. This study provides a assessment methods of the role of probiotics in the diet and may help optimize the use of probiotics as feed additives to replace antibiotics, thereby achieving the high safety and quality of production in fourfinger tetradactylum.