Abstract Flavonoids are important secondary metabolites that regulate plant growth and development and confer resistance against biotic and abiotic stress. As natural polyphenol substances, flavonoids determine the quality traits of commercial fruits, such as color, flavor, and nutrition. In the past few decades, research on the regulation of flavonoid biosynthesis in plants has made significant progress. However, a deep understanding of this aspect in flavonoid-rich horticultural crops is lacking. This review aims to systematically summarize the current knowledge in the regulation of flavonoid biosynthesis in fruits, including the transcriptional, post-transcriptional, epigenetic, and post-translational regulation mechanisms as well as the composite regulation cascades. Our analysis shows that direct transcriptional regulation involves the actions of different transcription factor families, such as MYB, WRKY, bZIP, AP2/ERF, and MADS, by directly targeting the key synthase genes in flavonoid biosynthetic pathway. Indirect regulation involves specific transcription factors and microRNAs that target the downstream regulators, as well as the regulation modules triggered for degradation of activators or repressors in response to environmental signals or plant hormones. In addition, epigenetic regulation, associated with methylation level in the gene promoter regions or the insertion or deletion of specific sequences therein, plays an important role in controlling anthocyanin accumulation. Based on the diverse regulation mechanisms of the flavonoid biosynthetic pathway, more molecular design targets can be applied in the future, facilitating the production of more stress-tolerant and quality-elevated crop varieties.