In order to explore the effect of biofilm culturing methods on start-up time of fluidized-sand biofilter (FSB) and structure of microbial community, understand the water purification mechanism of FSB, and apply it to Chinese sturgeon recirculating aquaculture system, the start-up time of FSB when using natural biofilm culturing method and inoculating probiotics was studied, and the change of structure of the microbial community in FSB at different times was analyzed with pyrosequencing method. The results showed that the start-up time of FSB when inoculating probiotics was significantly shorter than that when using natural biofilm culturing method. At the early period of biofilm culturing, the microbial species on the surface of filter material carrier was few, and the predominant bacteria were firmicutes. After the biofilm got mature, the diversity of bacterial community in the biofilter when using natural biofilm culturing method was significantly higher than that when inoculating probiotics, the community structure was more stable, and the proportion of functional bacteria was also higher than that of the inoculation method, such as Flavobacterium and purple nonsulfur bacteria, which indicated that the natural biofilm culturing method was more conducive to the stable operation of FSB.