Abstract: In order to explore the formation process, water purification effect, microbial structure and function of three types of biofloc in the water with Litopenaeus vannamei during hatchery period, the content of flocs and water quality index were measured, and Illumina MiSeq high-throughput sequencing technique was applied to compare the characte-ristics of biofloc in three groups with glucose, starch and sucrose, respectively. The results indicated that the biofloc formed in each group could effectively regulate water quality and reduce the levels of ammonia and nitrite. The concentrations of ammonia, nitrite and nitrate in the water samples from three experimental groups were significantly lower than those in the control group (P<0.05). The concentrations of ammonia, nitrite and nitrate in starch group were significantly higher than those in the glucose group and sucrose group (P<0.05). Moreover, the BFV in starch group was significantly lower than that in the glucose group (P<0.05), and the sucrose group possessed the highest BFV. The particle size of three groups was: sucrose group > glucose group > starch group. The quantity of 553, 515 and 542 OTUs was measured in the three groups respectively. Tthe microbial abundance index Chao1 and Shannon value implied the relationship in the three groups was: glucose group > sucrose group > starch group. Furthermore, regarding the phylum level, Proteobacteriae, Bacteroidetes and Planctomycetes were the dominant phylum in each group, and the proportion of three phylums in each group was 91.7%, 97.6% and 88.7%, respectively. However, in the genus level, Oceanicella was the dominant genus in glucose group, with the highest proportion of 18.4%, and the Muricauda and Cyclobacterium had the highest proportion in starch group, which were 9.8% and 5.9% respectively, and higher than those in glucose group and sucrose group. Rhodopirellula in sucrose group was 1.8% and 4.1% higher than that in the glucose group and starch group, respectively. Cell metabolism, genetic information processing and environmental information processing were detected in the three groups by Tax4Fun method. The gene function abundance in glucose group was higher than that in the sucrose and starch group. All of the results implied that biofloc in the breeding water with Litopenaeus vannamei could not only purify water quality, but also improve the microbial diversity, among which the glucose was the most effective carbon source. Nevertheless, biofloc technology is of essential to maintain the balance between water quality and water ecosystem.