To provide a basis for the application of Lactobacillus in the healthy aquaculture of tilapia, the effects of mixed culture of Lactobacillus as water additive on the environment of pond, and the health of intestine and gill of tilapia were studied. Mixed culture of Lactobacillus was added to the water of tilapia (Oreochromis niloticus) pond (control group: 0; experimental group: with a final concentration of 1.0×10⁴ cfu/mL) for ten weeks. The components of mixed culture of Lactobacillus were Lactobacillus casei and Lactobacillus acidophilus, with a 1:2.2 ratio. The water quality of pond, immune and antioxidant indexes of the intestine and gill of tilapia were monitored. High throughput sequencing technique (Illumina Miq) was used to compare the community structure of microbiota in water, sediment, intestine and gill mucosa of tilapia between the control and the experimental group. The results showed that the nitrate content in the water samples of the experimental group was lower than that of the control group. From the seventh week, and the nitrate content in the water samples of the experimental group was 72.68% (P< 0.05), 72.00% (P>0.05), 26.27% (P>0.05) and 21.46% (P>0.05) lower than that of the control group, respectively. The total nitrogen content in the water samples of the experimental group was 43.39% (P>0.05), 44.64% (P>0.05), 15.64% (P>0.05), 37.57% (P>0.05) and 34.49% (P>0.05) lower than that of the control group from the sixth week, respectively. The total phosphorus content in the water samples of the experimental group was 40.91% (P>0.05), 33.33% (P>0.05), 50.00% (P>0.05), 33.33% (P>0.05), 17.39% (P>0.05), 39.29% (P< 0.05) and 25.71% (P>0.05) lower than that of the control group from the fourth week, respectively. Compared with the control group, the activity of alkaline phosphatase (AKP) in the intestine of tilapia in the experimental group increased by 45.04% (P< 0.05), and the total superoxide dismutase (SOD) and AKP in the gill of tilapia in the experimental group increased by 58.26% (P< 0.05) and 60.99% (P< 0.05), respectively. Mixed culture of Lactobacillus influenced the community structure of microbiota of pond water, but did not affect that of sediment. Microbiota of intestine and gill of tilapia were affected by the probiotics, among which, the conditional pathogens, Acinetobacter and Pantoea were decreased, while the beneficial bacteria, Cetobacterium was increased. Using mixed culture of Lactobacillus as water additive could improve water quality, the immunity and antioxidant capacity of intestine and gill of tilapia, and regulate the community structure of microbiota of intestine and gill of tilapia.