Melatonin is a well-known molecule which possesses many beneficial effects on human health. Many agriculture products provide natural melatonin in the diet. Cherry is one such fruit as they are rich in melatonin. In order to understand the biological roles of melatonin in cherry fruit, melatonin synthesis and its changes over 24 hr period were systematically monitored both during their development and in the ripe cherries in two cultivars, ‘Hongdeng’ (Prunus avium L. cv. Hongdeng) and ‘Rainier’ (Prunus avium L. cv. Rainier). It was found that both darkness and oxidative stress induced melatonin synthesis, which led to dual melatonin synthetic peaks during a 24 hr period. The high levels of malondialdehyde induced by high temperature and high intensity light exposure were directly related to up-regulated melatonin production. A primary function of melatonin in cherry fruits is speculated to be as an antioxidant to protect the cherry from the oxidative stress. Importantly, plant tryptophan decaboxylase gene (PaTDC) was identified in cherry fruits. Our data shows that PaTDC expression is positively related to the melatonin production in the cherry. This provides additional information to suggest that tryptophan decaboxylase is a rate-limiting enzyme of melatonin synthesis in plants.