Nanospheres were prepared by different materials of nano-bamboo charcoal powder, zeolite powder, and aquaculture pond sediment in different ratios. It was then fermented with effective microorganisms (EM) active calcium liquid to synthesize the bioactive microbial nanospheres. These nanospheres were used to compare the purification effect of ammonium nitrogen (NH 4 + -N), total nitrogen (TN), and total phosphorus (TP) pollutants in aquaculture wastewater. The indoor simulation experiment was also conducted to compare the different dosage methods (one-time dosing without aeration, multiple dosing without aeration, and multiple dosing with aeration) of microbial nanospheres on the removal of organic matter (OM) and effect of the biodegradability (G value) in aquaculture sediment. The results obtained indicated that the purification effect was most remarkable when the mass ratio of nano-bamboo charcoal powder: zeolite powder: pond sediment was 10%: 15%: 75%, in which the maximum removal rate of NH 4 + -N, TN, and TP reached up to 84.86%, 52.15%, and 50.35%, respectively. Under the same microbial nanospheres amount, the effect of one-time addition on the removing of OM in sediment was not as effective as that of multiple dosing. After the 20th day, the removal rate of OM reached 25.99% in multiple dosing treatment and it was 35.58% higher than one-time dosing treatment. The OM content in sediment was reduced by 32.38% under the multiple dosing with aeration treatment. Multiple dosing of microbial nanospheres with aeration increased the G value of sediment about 337.0%. In situ experiment further indicated that the microbial nanospheres dosage with aeration had a good sediment bio-remediation effect, which is applicable to solve the problem of endogenous pollution in aquaculture ponds.leads to the ecological degradation and the serious diseases [3]. Therefore, repairing the eutrophic aquaculture environment requires not only the reduction of nitrogen, phosphorus, and organic matter in the aquaculture water, but also the removal of the sediment mud. In the aspect of aquaculture water purification, the leading technologies include biological filter treatment [4], artificial floating bed cultivation [5], artificial wetland [6], and so on. Due to the shortage of freshwater resources, the high density of breeding and a large amount of bait in the aquaculture industry as well as the present aquaculture water treatments still have problems, such as single treatment method adoption, high cost, and difficult operation [7]. In recent years, a new type of circulating water pond model is applied to reduce the nitrogen and phosphorus content in the aquaculture water, mainly relying on aquatic plants and microbes function [8]. Many studies [9][10][11] have found the composite microbial agents composed of various microbes could make the control effects of aquaculture water quality better. The EM population (containing more than 80 kinds of microbes, with photosynthetic bacteria, lactic acid bacteria, yeast, and actinomycetes as repr...