As an effective, safe and eco-friendly aquaculture mode, biofloc technology (BFT) is receiving more and more attention nowadays. So far, the impacts of BFT on perfomances of Litopenaeus vannamei regarding organic carbon sources, C/N ratio and culture density have been reported, which especially focusing on water quality and biofloc microflora. However, the effect of molasses on the aquatic microbiota during shrimp culture has not been systematically evaluated. In order to investigate the effects of molasses on shrimp growth and aquatic microbiota during culture of L. vannamei, high-throughput sequencing of 16S rDNA amplicons was used to compare the differences of bacterial community structure between the treatment group with molasses (C/N=16.0) and the control group (C/N=8.5) on the 4th, 13th and 34th days respectively, and the microflora function was predicted by PICRUSt2. The results showed that although molasses addition had no significant effects on body length and body weight of shrimp, the shrimp survival rate was significantly enhanced. Compared with the control group, the Shannon diversity and evenness indices of aquatic microbiota in treatment group increased significantly on the 4th day, while decreased markedly on the 13th and 34 th days. After adding molasses, the relative abundances of α-Proteobacteria and Actinobacteria in the treatment group were very significantly higher than those in the control group, whereas the abundances of Cyanobacteria and γ-Proteobacteria presented an opposite trend. The abundance of β-Proteobacteria was also significantly lower than that of the control group on the 4th and 34th days. Additionally, the abundance of Bacteroidetes was significantly higher and lower than that of the control group on the 4th and 13th days, respectively. At the family level, the relative abundance of Microbacteriaceae in the treatment group was significantly higher than that in the control group on the 4th, 13th, and 34th days. On the 4th day, the relative abundances of Rhodobacteraceae and Flavobacteriaceae were extremely significantly higher than those of the control group, while the abundances of Devosiaceae, Alcaligenaceae and Lewinellaceae exhibited conversely. On the 13th day, the abundances of Halieaceae and Cyclobacteriaceae were significantly lower than that of the control group. On the 34th day, the abundance of Demequinaceae was significantly higher than that of the control group. The abundances of dominant genera such as Gemmobacter and Demequina in the treatment group were significantly higher than those in the control group, while the abundances of some genera such as Haliea decreased significantly. The functional prediction of aquatic microflora showed that the top 20 items in the treatment group presented extremely significant metabolic function enrichment on the 34th day, and eight of these items, including glycolysis/gluconeogenesis, were highly enriched, which mainly act on carbohydrate or energy metabolism. The results indicated that the proper amount of molasses could significantly affect the relative abundances of dominant bacterial communities in rearing water of L. vannamei, optimize the microflora structure by enriching the functions of carbohydrate and energy metabolism pathways, and thus improve the survival rate of shrimp. This study provides key and practical references for application of molasses as an organic carbon source in shrimp biofloc aquaculture.