Lipid droplets (LDs) are dynamic organelles that are ubiquitous in most organisms, including animals, plants, protists and microorganisms. The core consists of neutral lipids, surrounded by a monolayer of phospholipids that is decorated by a specific set of proteins. As an important intracellular metabolic regulatory hub, lipid droplets play a crucial role in the maintenance of physiological homeostasis and the evolution of pathological processes. They store neutral lipids, which are mobilized for energy production during starvation or for membrane bio-synthesis and sequester fatty acids to protect against lipotoxicity. Clinically, the dysregulation of the function of lipid droplets is associated with a variety of diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD), obesity, diabetes mellitus type 2 (T2DM), neurodegenerative diseases and cancer. Researches on the important biological functions of lipid droplets as dynamic organelles and their association with multiple diseases have become a cutting-edge hotspot in the field of cell biology. In recent years, significant progress has been made in the field of lipid droplet biogenesis. First, the researchers have established a more refined framework for lipid droplet biogenesis and have systematically elucidated how the assembly of LDs occurs in the endoplasmic reticulum (ER). Triacylglycerols and sterol esters are synthesized between the inner and outer leaflets of the endoplasmic reticulum bilayer and subsequently nucleate and form neutral lipid lenses when they exceed the critical nucleation concentration (CNC). Then, under the synergistic effect of Seipin, fat storage-inducing transmembrane protein 2 (FIT2) and peroxisomal membrane protein Pex30, they are budded from the ER to form initial lipid droplets through changes in membrane curvature and surface tension induced by asymmetric distribution of phospholipids. Initial lipid droplets recruit lipid synthesizing enzymes through the ER-LD bridging structure to achieve local lipid synthesis and surface expansion, ultimately forming mature lipid droplets. Through biochemical and biophysical approaches, important features of lipid droplet biogenesis have been uncovered, highlighting the importance of the biophysical properties of the ER membrane and specific phospholipids in this process. Structural biology and proteomic methods have identified key factors including Seipin and FIT2 in LD biogenesis. This article provides a systematic review of the latest progress in LD biogenesis from molecular mechanism level. Furthermore, it explores the detailed aspects of LD nucleation, membrane budding and expansion in eukaryotic cells, especially the molecular mechanism of dynamic regulation of LD morphology by core factors such as Seipin and FIT2. Moreover, it discusses the mechanism and pathways of class I proteins and class II proteins targeting to LDs, compares the biogenesis of LDs with different neutral lipid core and elaborates the relevance of their specific regulators with diseases. Finally, we summarize critical unresolved questions in LD biogenesis, providing clear directions for future research and a systematic perspective for the deeper understanding of LD biogenesis and disease intervention strategies.