Abstract: Stevioside, also known as stevia, is a class of diterpenoids extracted from stevia with high sweetness, low calories, safety and non-toxicity, hypoglycemic, hypotensive, and other physiological characteristics. It is regarded as the "world's third natural glycogen," and it has been widely used in the food, pharmaceutical, daily-use chemicals, brewing, and other industries. However, extracts based on the traditional extraction process have ¹problems such as difficult purification, low extraction efficiency, and excessive consumption of solvents, which limit their value and application scope. The rapid development of synthetic biology provides a new green and efficient production mode for the production of plant-based natural products. To this end, the article reviews the research progress of steviol glycosides' biosynthesis based on the elucidation of their structure and physiological activities. It analyzes the biosynthetic pathways of steviol glycosides and the catalytic mechanisms of the key enzymes involved. The focus is on the mechanisms and approaches for biocatalytic synthesis using glycosyltransferases and the de novo synthesis of specific steviol glycosides by microorganisms. Additionally, it discusses the application of two biosynthetic methods in the production of high sweetness, low bitterness rebaudiosides A, D, and M. Based on the aforementioned research advances, the paper explores the main challenges currently faced in the biosynthesis of steviol glycosides and future research directions, aiming to provide theoretical insights for the biosynthesis of steviol glycosides.