Abstract: Linolenic acid is a functional polyunsaturated fatty acid, which is essential for human health and therefore is widely applied in many fields such as medicine and food. Compared with traditional animal and plant extraction, linolenic acid synthesized by microbial fermentation has significant advantages such as short production cycle, little environmental impact, and stable production efficiency. The key to microbial synthesis of linolenic acid lies in two aspects: one is to develop high-yielding strains, and another one is to optimize the fermentation strategy. This review systematically summarizes the microbial sources of linolenic acid, and deeply discusses the microbial synthesis pathway of linolenic acid, the characteristics and functions of key enzymes in the synthesis pathway, genetic engineering strategies to promote linolenic acid synthesis, and fermentation optimization methods of production strains. This review also analyzes the potential and challenges of using synthetic biology technology to improve microbial production of linolenic acid. By designing and constructing new biological systems, new possibilities for microbial production of linolenic acid are provided, which is helpful for better utilizing microorganisms in the future.