Surfactin, a representative cyclic lipopeptide biosurfactant produced by diverse microorganisms, is widely recognized as one of the most effective biosurfactants to date. Due to its remarkable surface and interfacial activities alongside significant biological properties, surfactin presents a sustainable complement to conventional surfactants and antibiotics. This review provides a comprehensive overview of recent advancements in the biosynthesis, structural characterization, molecular behaviors, and potential applications of surfactin. We first explore its microbial origins and biosynthetic pathways, followed by an in-depth discussion of laboratory-scale production process, encompassing microbial screening, fermentation process, purification, and structural identification using various characterization techniques. The structural diversity of surfactin, which varies in the length (C11-C18) and configuration of hydrophobic chain and the amino acid composition and methylation of heptapeptide, is systematically summarized. Furthermore, we highlight the colloidal and interfacial properties of surfactin, emphasizing the interplay between molecular structures, micelle formation, self-assembly behaviors, adsorption dynamics, and environmental factors. These exceptional properties position surfactin as an ideal candidate for exploitation in a wide range of applications, including enhanced oil recovery, environmental bioremediation, and innovations in the daily chemical and pharmaceutical fields.