(-)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus, which can be used as a raw material in cosmetics and has anti-inflammatory function. In this study, we designed six mutation sites of the (-)-α-bisabolol synthase BOS using the plmDCA algorithm. Among these, the F324Y mutation demonstrated exceptional performance, increasing the product yield by 73 %. We constructed a de novo (-)-α-bisabolol biosynthesis pathways through systematic synthetic biology strategies, including the enzyme design of BOS, selection of different linkers in fusion expression, and optimization of the mevalonate pathway, weakening the branching metabolic flow and multi-copy strategies, the yield of (-)-α-bisabolol was significantly increased, which was nearly 35-fold higher than that of the original strain (2.03 mg/L). The engineered strain was capable of producing 69.7 mg/L in shake flasks. To the best of our knowledge, this is the first report on the biosynthesis of (-)-α-bisabolol in Komagataella phaffii, implying this is a robust cell factory for sustainable production of other terpenoids.