A long-term reused culture solution of Spirulina platensis was added with nutrient salt for cultivation of itself. The effects of cell density, concentrations of phosphate and nitrate, and contents of biological components in it in its final cultivation on the growth of itself were examined. The compounds in the solution were classified in molecular weight by ultrafiltration membranes. Their inhibitory activity on the cultivation was measured, and molecular weight distribution of inhibitors analyzed, and their compositions were determined. The results showed that the long-term reused culture medium inhibited the cell growth of Spirulina platensis, although nutrition was complemented enough, the specific growth rate was decreased by 23%, compared with fresh culture medium, and the absorption rate of nitrate and phosphate was decreased by 36% and 37%, as the metabolism of nitrogen and phosphorus and the synthesis of intracellular proteins and chlorophyll of Spirulina platensis were inhibited. The content of intracellular proteins was 0.85 of that of fresh culture medium, and the content of chlorophyll 0.65. The auto-growth inhibitor of Spirulina platensis was extracellular polysaccharide with the molecular weights over 100 kDa.