Abstract: 【Objective】 To study the salt tolerance response mechanism of osmotic regulators of cotton seedlings under mixed salt and alkali stress. 【Methods】 Xinluzao 57 was taken as the research object, the NaCl, Na₂SO₄, Na₂SO₃, and Na₂CO₃ 4 salts were mixed in different proportions to simulate the stress conditions of mixed salt, and the alkaline salt proportion was gradually increased into A, B, C, D and E 5 groups and treated with different concentrations. 【Results】 The results showed that there was a significant correlation between osmoregulation substance and salt alkali ratio, salt alkali concentration (P < 0.01), and the interaction between salt alkali ratio and salt alkali concentration had a significant correlation with osmoregulation substance of cotton seedling leaves; proline content increased with the increase of salt alkali concentration. The content of proline in treatment B (NaCl, NaHCO₃) was significantly higher than those in other treatments with the increase of concentration, and the difference of proline content in treatment C (NaCl, Na₂SO₄, NaHCO₃, Na₂CO₃) was lower. At the concentration of 25 mmol/L, the proline content of leaves increased the most, and with the increase of concentration, the increase of proline content decreased significantly; the change trend of malondialdehyde and soluble sugar was similar to that of free proline, while the content of soluble protein increased with the increase of treatment concentration, but the increase was small; and the difference was not significant. 【Conclusion】 The results showed that there is a significant correlation between osmotic adjustment substances and salt alkali ratio and concentration (P < 0.01), and the interaction between salt alkali ratio and salt alkali concentration has a significant correlation on osmotic adjustment substances of cotton seedling leaves. Under the same mixed salt treatments, the contents of proline, soluble sugar, malondialdehyde and free proline increase with the increase of salt concentrations. The content of soluble protein increases little with the increase of concentration, but the difference is significant in different saline alkali ratios of the same salt concentration, which further indicates that the soluble protein content of cotton seedling leaves under mixed salt alkali stress is the main osmotic adjustment substance to evaluate the salt tolerance of cotton.