Natural sweeteners have attracted widespread attention because they are eco-friendly, healthy, low in calories, and tasty. The demand for natural sweeteners is increasing together with the popularity of green, low-carbon, sustainable development. With the development of synthetic biology, microbial cell factories have emerged as an effective method to produce large amounts of natural sweeteners. This technology has significantly progressed in recent years. This review summarizes the pathways and the enzymes related to the biosynthesis of natural sweeteners, such as mogrosides, steviol glycosides, glycyrrhizin, glycyrrhetinic acid, phlorizin, trilobatin, erythritol, sorbitol, mannitol, thaumatin, monellin, and brazzein. Moreover, it focuses on the research about the microbial production of these natural sweeteners using synthetic biology methods, aiming to provide a reference for future research on the production of natural sweeteners.