||Adaptive laboratory evolution (ALE) is an increasingly popular technique, which allows screening the strains with excellent phenotype through accumulative positive changes. However, little strategy based on ALE has been previously reported about improving carotenoids yield in S. cerevisiae. Herein, a new ethanol-induced ALE was successfully applied to boost carotenoids accumulation in a carotenoid producer BL03-D-4. Following the novel ALE strategy, an evolved strain M3 was obtained through adaptive evolution with a 5.1-fold increase in carotenoids yield from 3.9 mg/g CDW (cell dry weight) to 19.7 mg/g CDW in the YPD medium. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of PFK1 was revealed as the cause of increased carotenoids yield. Subsequent transcriptome analysis was conducted to reveal the potential mechanisms for improved carotenoids yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study demonstrates that ethanol is a powerful stress to obtain high carotenoids accumulation strains and provides PFK1 as a new target for improving carotenoids yield in S. cerevisiae.