Abstract: The suitable fermentation strains were screened, and the waste residue and waste water of chestnut were used as fermentation base. The content of live lactic acid bacteria and the content of yeasts after fermentation were used as indicators to investigate the effects of different fermentation conditions on the viable count in the fermentation broth. Factor experiment and L₉ (3⁴) orthogonal test were used to determine the process of preparing aquatic probiotics from processing waste. The results showed that Lactobacillus plantarum (BL191) and Saccharomyces cerevisiae (SC187) were suitable for the fermentation of probiotics. The optimum mixed fermentation process conditions mixing ratio of waste residue and waste water of chestnuts1:2 (g/mL), mixing ratio of BL191 and SC187 3:7 (g/g), inoculum volume 12%, fermentation temperature 34℃, fermentation time 48 h. The prepared aquatic probiotics had a content of BL191 bacteria of 33.9×10⁸ CFU/g, and the content of SC187 bacteria was 15.9×10⁸CFU/g. It was rich in nutrients, and the total amino acid amount was 1.8 times higher than that before fermentation. The essential amino acid was increased by more than 48%. This study would be helpful to solve the environmental pollution caused by water chestnut during the processing, and it also provide a theoretical basis for aquaculture to reduce the abuse of antibiotics.