Farnesene is increasingly used in industry, agriculture, and other fields due to its unique and excellent properties, necessitating its efficient synthesis. Microbial synthesis is an ideal farnesene production method. Recently, researchers have used several strategies to optimize the production performance of microorganisms. This review summarized these strategies, including regulation of farnesene synthesis pathways, and proposed some emerging tools and methods in stain engineering. Meanwhile, new farnesene biosynthetic pathways and effective farnesene production from cheap or waste substrates were emphatically introduced. Finally, future farnesene biosynthesis challenges were discussed.