Melatonin (N-acetyl-5-methoxytryptamine) is a molecule implicated in multiple biological functions. Its level decreases with age, and the intake of foods rich in melatonin has been considered an exogenous source of this important agent. Orange is a natural source of melatonin. Melatonin synthesis occurs during alcoholic fermentation of grapes, malt and pomegranate. The amino acid tryptophan is the precursor of all 5-methoxytryptamines. Indeed, melatonin appears in a shorter time in wines when tryptophan is added before fermentation. The aim of the study was to measure melatonin content during alcoholic fermentation of orange juice and to evaluate the role of the precursor tryptophan. Identification and quantification of melatonin during the alcoholic fermentation of orange juice was carried out by UHPLC-QqQ-MS/MS. Melatonin significantly increased throughout fermentation from day 0 (3.15 ng/mL) until day 15 (21.80 ng/mL) reaching larger amounts with respect to other foods. Melatonin isomer was also analysed, but its content remained stable ranging from 11.59 to 14.18 ng/mL. The enhancement of melatonin occurred mainly in the soluble fraction. Tryptophan levels significantly dropped from 13.80 mg/L (day 0) up to 3.19 mg/L (day 15) during fermentation. Melatonin was inversely and significantly correlated with tryptophan (r = 0.907). Therefore, the enhancement in melatonin could be due to both the occurrence of tryptophan and the new synthesis by yeast. In summary, the enhancement of melatonin in novel fermented orange beverage would improve the health benefits of orange juice by increasing this bioactive compound.