Mulberry fruit is in increasingly demand as a healthy natural product. Research into the compositional characteristics of mulberries at different stages of ripening could help maximise their use and minimise wastage, but knowledge in this area is limited. This study investigated the physicochemical and transcriptional expression profiles of mulberries at different maturity stages. The results showed that the nutrients of mulberries generally accumulate during ripening. Ripe mulberries contained high levels of total sugars (129.88 ± 1.09 mg/g), total phenolics (3.08 ± 0.16 mg/g), and anthocyanin compounds, making them a good source of dietary supplements and medicines for the population. Interestingly, unripe mulberries had a high content of minerals and organic acids, showing their potential value as a derivative product. Especially, the highest content of potassium (2237.00 ± 86.56 μg/g), magnesium (707.04 ± 24.29 μg/g), calcium (2371.20 ± 364.09 μg/g), phosphorus (545.90 ± 3.13 μg/g), citric acid (45.96 ± 0.65 mg/g) were detected in the unripe mulberry. Transcriptome sequencing revealed high transcript levels of UDP-glucosyltransferase 29 and bHLH 151 in ripe mulberries. KEGG enrichment analysis indicated that the flavonoid biosynthetic pathway is a key pathway throughout the ripening process, and the high expression levels of CHS, CHI, F3H, F3′H, DFR, and ANS may contribute to flavonoid accumulation. By understanding and exploiting the characteristics of mulberries at different stages of ripening, it will help to efficiently develop nutrient-enriched mulberry derived products.