The mulberry leaf (the dried leaves of Morus alba L.) is a widely used traditional Chinese medicinal herb. With a growth cycle of approximately eight months, determining the optimal harvest period is essential for ensuring its medicinal quality. Although traditional pharmacopoeias consider the superior quality of “post-frost mulberry leaves”, the underlying material basis and molecular regulatory mechanisms remain unclear. This study aims to systematically characterize the dynamic accumulation patterns of chemical constituents and elucidate the key components and regulatory networks responsible for the enhanced quality of “post-frost mulberry leaves”.