Abstarct: Salinity stress is one of the most important abiotic stresses in Iran and the world, which leads to a significant reduction in crop yield each year. Production and development of salinity tolerance cultivars is one of the most important methods of reducing damage caused by salinity stress. Development of tolerance cultivars requires evaluation and recognition of morphological, biochemical and molecular mechanisms of plants sensitive and resistant to stress, because it is vital in promoting human knowledge and identifying plant mechanisms to stress. The use of various stimulants to provide tolerance to crops has become an important and attractive research topic in recent years. The aim of this study was to investigate the effects of different levels of melatonin and cold plasma on physiological and biochemical characteristics as well as the exxpression of a number of genes involved in the biosynthesis pathway of diosgenin in fenugreek. The treatments of this study included salinity of 200 mM, different levels of melatonin (0, 30 and 60 ppm) and cold plasma voltages (3000 and 4000 volts) and various times after plasma exposure (1, 2 and 4 minutes). The results of this study showed that the application of different levels of melatonin and cold plasma treatments (both under normal conditions and under salinity stress) significantly led to positive changes in the traits evaluated. Melatonin (60 ppm) and cold plasma (especially 3000 volts and 2 min) prevented chlorophyll and carotenoid degradation and increased electrolyte leakage index and malondialdehyde content under salinity stress condition. Also, the application of these two treatments effectively led to an increase in the activity of antioxidant and non-antioxidant enzymes in fenugreek under salinity stress, thus enhancing the strength of the plant's defense system. In addition, the application of different levels of melatonin and cold plasma effectively prevented auxin depletion under salinity stress condition. The exxpression of SQS, SEP, CAS, SSR, SMT, BGL genes and diosgenin content increased 13, 15.4, 22.5, 21.66, 12, 19 and 6.5 times compared to the control, respectively. The findings of this study increased our understanding of the melatonin role and cold plasma in imparting fenugreek tolerance through changes in plant pigment mextabolism, antioxidant and non-antioxidant enzymes, cellular homeostasis and secondary mextabolites, as well as changes in diosgenin content in fenugreek under salinity stress.