Hypertrophic scar (HS) is a disease with excessive skin fibrosis and collagen disorder, which is generally caused by abnormal wound repair process after burn and trauma. Although intralesional injection of 5-fluorouracil (5-Fu) has been used in clinical treatment of HS, the patients' compliance of injection treatment is poor. In this study, a double-layer dissolution microneedle (MN) containing asiaticoside (AS) and 5-Fu was designed for the treatment of HS. Biological macromolecules materials affected the formability, drug release, and hardness of MNs. Therefore, several types of biomacromolecules, including hyaluronic acid (HA), chitosan, and sodium alginate, which could be used to prepare MNs, underwent prescription optimization experiments and the optimized MN prescriptions were obtained. In vitro characterization showed that the MN was sufficient to deliver drugs through the skin. Animal in vivo experiments showed that AS and 5-Fu can synergistically treat HS, significantly reduce the abnormal proliferation of fibroblasts and collagen fiber deposition, and down-regulated collagen I (Col I) and transforming growth factor-β