BACKGROUND Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production. RESULTS We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica. CONCLUSIONS Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.