Alfalfa (Medicago sativa L.) is the most cultivated forage legume in Iran that productivity and quality of that are limited by salt stress. Melatonin as an indole amine was used for this study, with different concentrations of melatonin (0.1, 10 and 15 μM) under in vitro salt stress (150 and 200 mM NaCl) of alfalfa seedlings which by increasing salinity, growth rate as well as antioxidant power and synthesis of antioxidant compounds decreased. Growth parameters and defense system such as antioxidant enzymes were reduced in plants under salt stress. Exogenous utilization of melatonin in alfalfa significantly elevated fresh and dry weight, shoot length, photosynthetic pigments and carotenoid content. Melatonin application was augmented phenolic compounds such as total phenol, flavonoids and also antioxidant enzymes, phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL). According to our findings, Melatonin could be an excellent candidate for increasing salt resistance, by antioxidant capacity, scavenging ROS, reducing oxidative damage, augmented of non-enzymatic antioxidant such as phenolic compounds and on the other hand increasing the photosynthetic capacity of the plant by preserving photosynthetic pigments in alfalfa seedlings.