In flavonoid biosynthesis, the phenylpropanoid branch has received increasing attention, but little attention has been paid to the fatty acid β-oxidation branch involved in providing the necessary malonyl-Coenzyme A (CoA) moieties of flavonoids. Here, we conducted a qualitative and quantitative metabolomic analysis of nine apple cultivars with different flavonoid contents by using Orbitrap high-resolution and triple quadrupole (QQQ) mass spectrometry. The results showed that flavonoid-rich apple cultivars had higher accumulation of several major flavonoids (such as phloridzin, epicatechin, and proanthocyanin B1) and other compounds related to fatty acid β-oxidation (such as 18-carbon unsaturated fatty acids, jasmonic acid) compared with those with low flavonoid contents. Then transcriptomic analysis revealed upregulation of genes involved in fatty acid β-oxidation (such as ACX, ECH, and KAT), jasmonic acid biosynthesis (such as LOX, AOC, and OPR), and flavonoid biosynthesis (such as CHS, CHI, F3H, ANS, ANR, and several FLSs) in flavonoid-rich apple cultivars. Finally, apple fruits at the coloring stage were treated with 100 and 200 μM etomoxir (an effective inhibitor of fatty acid β-oxidation). The results showed that etomoxir significantly decreased the fruit color, the flavonoid content, and the expression of flavonoid-related genes in a dose-dependent manner. These results highlight the important role of fatty acid β-oxidation in apple flavonoid biosynthesis and provide a theoretical foundation for improving flavonoid biosynthesis from the perspective of malonyl-CoA instead of phenylpropanoid.