Abstract: Salt stress is one of the factors that seriously affect the growth and development of cotton roots. Melatonin (MT) has the functions of regulating plant growth and antioxidation, nitric oxide (NO) is a signal molecule that regulates plant growth, Both play important roles in plant stress resistance. To clarify the regulatory effects of MT and NO on root growth and physiology of cotton seedlings under salt stress, this study used 'Jinongda 36' as the experimental material and employed hydroponic culture, by externally applying MT, SNP (NO donor), as well as combinations of MT and SNP, and MT and c-PTIO (NO scavenger), the study analyzed seedling root morphology, antioxidant enzymes, osmotic regulators, and NO content. The results showed that salt stress significantly reduced the main root length, number of first-order lateral roots, average root diameter, total root length, total root volume and dry weight of the roots of cotton seedlings, decreased superoxide dismutase (SOD) activity, soluble sugar content, soluble protein content and K⁺ content, while increasing peroxidase (POD) activity, malondialdehyde (MDA) content, Na⁺ content and NO content. Exogenous MT or SNP could effectively alleviate the effects of salt stress, increase endogenous NO content, enhance the activities of SOD and POD, reduce the content of MDA, increase the contents of soluble sugar and soluble protein, decrease the content of Na⁺, increase the content of K⁺, and promote root growth. Moreover, the combined treatment of MT and SNP had a better regulatory effect on the roots under salt stress compared with the single treatments. In addition, c-PTIO could significantly inhibit the alleviation effect of exogenous MT on salt stress. Therefore, the combined application of exogenous MT and NO can effectively reduce the impact of salt stress on the roots of cotton seedlings, and the regulatory effect of exogenous MT on the roots of cotton seedlings under salt stress depends on the production of NO.