Abstract: Dissolving microneedles (DMNs) are promising transdermal drug delivery systems due to their unique properties, including controlled drug delivery, tunability, and ease of self-administration for patients. In this review, great efforts were made to summarize the advances of microneedles including classification, materials for microneedles, and the latest fabrication methods. Importantly, a variety of representative biomedical applications of DMNs such as small molecules, peptides, proteins, and nucleic acids delivery were highlighted. At last, conclusion and future perspective for the development of advanced DMNs in biomedical fields were systematically discussed. Taken together, as an emerging tool, DMNs have shown profound promise for biomedical applications, providing reference for the research and development of more accurate and intelligent dissolving microneedles in transdermal drug delivery system.