<p>Melatonin is one of the important endogenous signaling molecules for regulating plant growth and development, and various abiotic stress responses. UV-B is an intrinsic component of sunlight. Plants are particularly vulnerable to UV-B radiation compared to other abiotic stressors due to their sessile growth patterns. Previous studies reported that melatonin played a key role in <i>Arabidopsis</i> UV-B signaling responses and UV-B resistance. However, there are still few reports on the biological effects of melatonin in leguminous plants responses to UV-B. We have selected alfalfa pretreated with exogenous melatonin, coumarin, or 5-hydroxytryptamine as biological materials, and the interactive effects between melatonin metabolisms in plant and UV-B radiation will be investigated by testing hypocotyl elongation, intracellular reactive oxygen species (ROS) accumulation, physiochemical reaction, antioxidant defense, and its related gene expression. The results have showed that UV-B radiation significantly inhibited hypocotyl elongation, altered leaf phenotype and photosynthetic parameters, induced ROS levels and cell membrane damage, decreased antioxidant enzyme activities except for POD (Peroxidase), and downregulated related gene expression. However, exogenous melatonin efficiently repaired detrimental effects by UV-B radiation through activating antioxidant defense and enhancing photosynthetic efficiency. We have also found that 5-hydroxytryptamine treatment displayed the same biological effects via increasing endogenous melatonin. However, coumarin application had no obvious improvements. These data will provide experimental foundation for further understanding legume responses to UV-B and its molecular mechanisms, and also fill the research gaps in this field.</p>