Melatonin (MT) can improve plant resistance and fruit quality. The mechanism by which MT affects soluble sugar and organic acids accumulation in drupe fruits is not clear. In this study, 100 µmol/L MT was sprayed on the leaves of plum trees at the second stage of rapid fruit expansion (90 and 97 d after flowering), and the effects of MT on plum fruit quality and its effects on the soluble sugar-organic acid metabolism were investigated. At 28 d after MT treatment (at maturity), the longitudinal diameter, fruit weight, and vitamin C content of plum fruits were increased by 5.05%, 12.93%, and 56.09%, respectively, compared to the control. MT caused significant increase in the total soluble solids content and decreased the titratable acid content. MT increased the contents of total soluble sugar, sucrose, sorbitol, and citric acid after 21 and 28 days of treatment, while decreasing the contents of fructose, malic acid, quinic acid, and tartaric acid after 28 days of treatment. Additionally, MT increased the activities of sucrose synthase (catabolism direction), sucrose phosphate synthase, glucokinase, fructokinase, sorbitol oxidase, and NADP
+
-malic enzyme, and decreased the activities of soluble acid converting enzyme, cell wall insoluble converting enzyme, NAD
+
-sorbitol dehydrogenase, and NAD
+
-malic dehydrogenase after 21 or 28 days of treatment. Moreover, the differentially expressed genes (DEGs) after 21 and 28 days of treatment were accelerated starch and sucrose metabolism, galactose metabolism, fructose and mannose metabolism, as well as glycolysis, gluconeogenesis, and pentose phosphate metabolism pathways. In conclusion, exogenous MT increases soluble sugar content and decreases organic acid content in plum fruits by regulating various soluble sugar-organic acid metabolic pathways, thereby improving the fruit quality.