To determine the best change frequency of substrates in the sea cucumber seedling cultivation system in summer, the relationship between the substrate change frequency and growth of sea cucumbers, and the microflora structure on the substrate, was investigated using an experimental ecology method, a traditional bacterial culture method and 16S rDNA sequence analysis. Five different substrate change frequency groups (10 d, 20 d, 30 d, 40 d, 50 d) were set and termed CF₁₀, CF₂₀, CF₃₀, CF40 and CF₅₀. Judged by the weight gain of the whole cultivation tank and the main body weight, the group achieved the best growth rate, followed by the CF₃₀ group. The CF₅₀ group achieved the highest death rate, which caused a decrease in the weight of the whole tank. The specific growth rate and the survival rate of the CF₂₀ group were (5.986±0.135)%/d and (95.231±0.265)%, respectively, which were significantly higher than the other groups (+-N), nitrite (NO₂^--N) and chemical oxygen demand (COD) of the pond water increased along with the decrease in the change frequency of substrates, reaching 0.53 mg/L, 0.28 mg/L and 0.18 mg/L on the 50th day, respectively. In terms of the concentrations of heterotrophic bacteria and vibrios in the pond water and on the substrate, the concentrations of heterotrophic bacteria and vibrios in the pond water did not change significantly with the change frequency of substrates; however, the concentrations of heterotrophic bacteria and vibrios on the substrate increased with decreasing change frequency of substrates, reaching 1.38×10⁵ cfu/cm² and 1.5×10⁴ cfu/cm² on the 50th day in the CF₅₀ group, respectively. Bacterial identification showed that the dominant bacteria on the substrates were Staphylococcus equorum, Vibrio parahaemolyticus, of which is an etiological pathogen for ; therefore, it might have a close relationship with the occurrence of skin ulcerative syndrome in the CF₅₀ group. Harmful animals, such as , compete for living space and food, causing slow growth of sea cucumbers. These animals emerged when the substrates had not been changed for a long time. The above results indicated that the optimal substrate change was once every 20 d in summer (July to September). Taking the production cost in consideration, it was suggested that the substrate should normally be changed once between 20 d and 30 d. These results will help to optimize breeding processes and health management for sea cucumbers.