Temporal changes in total flavonoid content (TFC), composition, free radical scavenging activity and metabolism-related gene expression of three Prunus humilis cultivars with distinctively different fruit colors were investigated in this study. The highest fruit TFCs of all three cultivars were observed at the initial sampling stage (young-fruit stage, YFS), which then declined gradually until fruit ripening. The dark-red-fruited cultivar ‘Jinou 1’ had the highest TFC, followed by the yellow-red-fruited cultivar ‘Nongda 3’ and the yellow-fruited cultivar ‘Nongda 5’. Thirteen flavonoid compounds were found in the three cultivars by using high-performance liquid chromatography (HPLC), and the content of most flavonoid compounds gradually decreased throughout the fruit-ripening process, with the exception of cyanidin-3-O-glucoside (C3G). C3G, as the main anthocyanin in P. humilis fruits, increased drastically during the fruit-coloring process of cultivars ‘Jinou 1’ and ‘Nongda 3’, while it was not detected in the developing fruits of cultivar ‘Nongda 5’. The antioxidant activity assay (DPPH, FRAP and ABTS) revealed that fruits of all three cultivars at YFS also had the highest antioxidant activities, and cultivar ‘Jinou 1’ had the highest antioxidant activities. Correlation analysis revealed that the antioxidant activities were significantly positively correlated with the TFCs and contents of the main compounds such as catechin, proanthocyanidin B1 and phloretin-2′,4-O-diglucoside (p < 0.01). Moreover, gene expression analysis showed that the flavonoid biosynthetic genes had different expression patterns in the three cultivars. The expression levels of ChCHS, ChCHI, ChF3H, ChDFR, ChLDOX and ChUFGT increased gradually with fruit ripening in cultivar ‘Jinou 1’, while all flavonoid-related genes in cultivar ‘Nongda 5’ decreased gradually during fruit development. The results from our study could significantly contribute to the deeper understanding of flavonoid accumulation mechanisms in P. humilis fruits and also help facilitate the targeted cultivar development and the utilization as a functional food of this fruit species.