The current study evaluated the effect of a carbohydrate addition strategy set at two biofloc levels on the establishment of nitrification in new biofloc technology aquaculture systems. Three carbohydrate addition strategies were chosen as follows: one-time glucose addition with 20:1 of the input C/N ratio, glucose additions at a C/N ratio of 20:1 when total ammonia nitrogen (TAN) > 2 mg/L, and only providing polycaprolactone (PCL) as carbohydrate with no glucose. Each strategy was supplied at two biofloc levels (300 mg/L and 800 mg/L). Significant differences of dissolved organic carbon (DOC) to total ammonia nitrogen (TAN) (DOC/TAN) in the water were observed among the six groups. The development of TAN, nitrite nitrogen (NO2−-N) and nitrate nitrogen (NO3−-N) in the groups with the same biofloc level did not differ significantly (p > .05) among the groups with the different carbohydrate addition strategies. Peak concentrations of TAN, NO2−-N and NO3−-N in groups with 800 mg/L biofloc were significantly higher than those of groups with 300 mg/L biofloc. The peak concentrations of TAN, NO2−-N and NO3−-N in the groups with PCL as carbohydrate were significantly lower than those of the other two addition strategies. The carbohydrate addition strategies influenced the bacterial community composition at the phylum level, which varied significantly among the groups, and the differences were more obvious compared to those of the different floc levels. The relative abundance of the Nitrospirae phylum and genus Nitrosomonadaceae-norank were notably low in all three groups. The bacteria found in high abundance had the following characteristics: they degraded organic substrates, had roles in nitrification or denitrification, and had the ability to attach to surfaces and co-aggregate. The strategy of one-time carbohydrate addition at a C/N ratio maintained at 20:1 had the best performance for nitrification. Multiple glucose addition at a C/N ratio of 20 when TAN >2 mg/L extended the nitrification establishment. Only the use of PCL as the sole carbohydrate showed a good performance of nitrification, assimilation and denitrification.