Glycopeptide antibiotics have potent inhibition of gram positive bacteria. They are also as the last line of defense for dealing with the serious infections caused by such bacteria. As the drug-resistant pathogens spread, the application of glycopeptide antibiotics becomes more and more restricted. In this review, the structural characteristic of glycopeptide antibiotics and their structure-bioactivity relationship, the mechanism of biological activity and resistance for pathogenic bacteria of glycopeptide antibiotics were summarized. Furthermore, the biosynthetic mechanism and targeted generation of novel glycopeptide antibiotics by synthetic biology strategy for clinical application was also discussed.?Finally, the problems in the glycopeptide antibiotic application were prospected.