Melatonin is a well-known bioactive molecule with an array of health-promoting properties. Here, we detected the physiological function of melatonin in transgenic switchgrass overexpressing the homologous sheep arylalkylamine N-acetyltransferase and hydroxyindole O-methyltransferase genes, which catalyze the last two steps of melatonin synthesis. Compared to the wild-type (WT) and transgenic control (EV, expressing the empty vector only) plants, the transgenic switchgrass showed higher melatonin levels. Melatonin was detected in almost all switchgrass tissues, and relatively higher levels were detected in the roots and stems. Besides, melatonin showed diurnal or circadian rhythms in switchgrass similar to that in other species. Furthermore, we also found that melatonin positively affected switchgrass growth, flowering and salt tolerance. The genes related to flowering (APL3, SL1, FT1, FLP3, MADS6 and MADS15) and salt stress resistance (PvNHX1) in transgenic switchgrass exhibited a different expression profiles when compared to the control plants. Our study provided valuable findings that melatonin functions as a promoter in the regulation of switchgrass growth, flowering and salt tolerance.