The experiment was conducted to study the effects of different carbohydrate-to-lipid ratio diets on growth, physiological and biochemical indices, gut microbiota composition and disease resistance of juvenile GIFT tilapia (Oreochromis niloticus). Fish with an average body weight of 27g were selected as the research object. Five isonitrogenous and isoenergetic diets with different carbohydrate-to-lipid ratios were used respectively: 44.2/4(D1), 39.6/6(D2), 35/8(D3), 30.4/10(D4), 25.8/12(D5) and fed for 10 weeks. Nutritional and biochemical analysis methods combined with high-throughput sequencing technology were used to determine the indices. The results showed that the changed C/L ratio had significant effect on the growth of GIFT tilapia in the early feeding stage of 2 weeks, both the growth effects of low- C/L and high- C/L ratio groups were not good. While in 4-10 weeks, there was no significant difference among all groups (P > 0.05). At 10 weeks, low C/L ratio-D4 was significantly higher than high C/L ratio-D1 in viscera/body ratio ( P < 0.05), while whose serum urea and disease resistance were lower than D1 ( P < 0.05). Moreover, mesenteric index of D4 was higher than high C/L ratios D1 and D2 ( P < 0.05), while whose whole-body crude protein was significantly lower than D1 and D2 ( P < 0.05). On the other hand, the hepatosomatic of D1 was higher than that of low C/L ratio-D5 ( P < 0.05), and the low-density lipoprotein cholesterol and glucose tolerance of D1 were higher than those of other groups ( P < 0.05). The species composition and heat map showed that D4 was separated from the other groups. In terms of phylum heatmap, Chlamydia, Chloroflexi and Actinomycetes of D4 were higher than other groups (especially D2 and D1), while its firmicutes were lower than other groups (especially D2). Further at genus level, Phreatobacter of D4 was significantly higher than that of other groups, while Lactobacillus and Bifidobacterium in D4 was significantly lower than that of other groups (except D3). In conclusion, GIFT tilapia has strong adaptability and utilization ability to different C/L ratio diets. Under the experimental conditions, although 39.6/6(D2) diet group had no significant difference with other groups in growth, its whole-body crude protein content was the highest among groups, and the composition of gut microbiota was also superior to other groups, which was an appropriate C/L ratio for GIFT tilapia.