The application of melatonin (MT) has been shown to improve the quality during the storage of fruits and vegetables. The primary objective of this study is to investigate the effects of MT on the quality of fresh-cut Gastrodia elata during low temperature (4°C) storage. The results indicate that MT treatment could suppress the respiratory rate and malondialdehyde content of fresh-cut G. elata, while also slowing down the decline in total acidity and total soluble solids. The treatment with MT reduced the superoxide anions and hydrogen peroxide production, inhibited the activity and expression of peroxidase and polyphenol oxidase. Additionally, it led to increased activity and expression of antioxidant-related enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase, while also resulted in elevated levels of ascorbic acid and glutathione. Furthermore, the treatment with MT induced an increase in total phenolic and flavonoid content of fresh-cut G. elata and enhanced the activity and expression of key enzymes involved in phenylpropanoid pathway (phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, 4-coumarate: CoA ligase). In summary, MT enhances the antioxidant capacity by activating both ROS metabolism and phenylpropanoid pathway, thus maintaining the quality of fresh cut G. elata.