In order to study the suitable carbon source required by the biofloc formation in Litopenaeus vannamei culture tank, 3 groups added with 3 carbon sources (glucose group, starch group and sucrose group) were designed. Each treatment group set up 3 replicates. The culture period was 20 d. The experiment analyzed influences of adding different carbon sources on biofloc formation, nutritional ingredients, microflora and water quality indexes. The findings showed that when carbon additive amount was 80% of feeding, the biofloc formation could effectively regulate water quality and reduce ammonia nitrogen and nitrite nitrogen level in water body. The concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen concentrations in three carbon sources addition groups were significantly lower than those of control group. The ammonia nitrogen, nitrite nitrogen and nitrate nitrogen concentrations in the starch group were significantly higher than those of the glucose group and sucrose group. The survival rates of the glucose group, starch group, sucrose group and control group were 72.9%, 54.2%, 69.8% and 44.3% respectively. The biofloc settling volume (BFV) in the starch group was significantly lower than the glucose group, while BFV in the sucrose group was the highest. After 13−15 d, three groups tended to be stable. Crude protein contents in glucose group and sucrose group were significantly higher than that of the starch group. There was no significant difference in the glucose group and sucrose group. The essential amino acids-histidine, arginine, methionine and the nonessential amino acids-aspartate, glusate, alanine in the starch group was lower than the glucose group and sucrose group. The essential amino acid index (EAAI) in the glucose group, starch group and sucrose group was calculated at 0.93, 0.89 and 0.92, respectively. The high-throughput sequencing results showed that there were more than 18 species of bacterial community at the phylum level. Proteobacteria and Bacterioidetes in three types of Biofloc had the highest ratio. In the starch group, Bacteriodetes content was significantly higher than other groups, while Planctomycetes and Actinobacteria contents in the sucrose group were obviously higher than the glucose group and starch group. Therefore, different additions of carbon sources in the L. vannamei culture tank affected the biofloc formation, nutritional ingredients, microbial community structure and diversities and improved water quality in different degrees. With the EAAI as the evaluation index, glucose and sucrose can be used as the suitable carbon source choices for L. vannamei culture pond.