The trisaccharide, 2′-fucosyllactose (Fucα1-2Galβ1-4Glc; 2′-FL), is the most abundant oligosaccharide in human milk. It has numerous significant biological properties including prebiotics, antibacterial, antiviral, and immunomodulating effects, and has been approved as “generally recognized as safe” (GRAS) by the Food and Drug Administration (FDA) and as a novel food (NF) by the European Food Safety Authority (EFSA). 2′-FL not only serves as a food ingredient added in infant formula, but also as a dietary supplement and medical food material in food bioprocesses. There is considerable commercial interest in 2′-FL for its irreplaceable nutritional applications. This review aims at systematically elaborating key functional properties of 2′-FL as well as its applications. In addition, several approaches for 2′-FL production are described in this review, including chemical, chemo-enzymatical, and cell factory approaches, and the pivotal research results also have been summarized. With the rapid development of metabolic engineering and synthetic biology strategies, using the engineered cell factory for 2′-FL large-scale production might be a promising approach. From an economic and safety point of view, microbial selection for cell factory engineering in 2′-FL bioprocess also should be taken into consideration.