Abstract: Human milk oligosaccharides (HMOs), as unique bioactive components in breast milk, have gained great scientific interest in recent years due to their potential beneficial mechanisms for infant health. To systematically elucidate the research status, hotspots, and developmental trends in the field of HMOs, a multidimensional bibliometric analysis of 3282 HMOs-related research articles indexed in the Web of Science Core Collection database was performed from 2000 to present. Firstly, quantitative analysis of the annual publication trends and journal distribution patterns were carried out. Then the visualization maps of country, institution, author, and keyword were generated to identify core research nations, significant research organizations, high-productivity author groups, and research hotspots through CiteSpace. The analytical results demonstrated that the field of HMOs was undergoing exponential growth, and the research institutions and scientific researchers in the United States occupied a core position. It was worth noting that the domestic research on HMOs was showing a rapid growth trend and Jiangnan University has driven significant research advances. Current research hotspots mainly focused on two directions, including the regulatory mechanism of HMOs interactions with intestinal microbiota, and the development of biosynthetic technologies for HMOs using Escherichia coli cell factories. Compared to chemical synthesis, enzymatic and microbial synthesis were widely recognized as more promising strategies for HMOs synthesis owing to their sustainability, efficiency, and high specificity. Given the surging demand for HMOs in infant formulas and functional foods along with continuous advances in synthetic biology, the enhancement of yield and synthesis efficiency of HMOs was urgent to overcome this technical bottleneck. Accordingly, biosynthesis methodologies for HMOs production were comprehensively categorized and evaluated, aiming to provide valuable insights to support industrialization and guide future developments in this field.