•De novo synthesis of longifolene was carried out for the first time in yeast cell factory.•Constitutive Promoters were characterized.•Screening of potential longifolene synthase for the production of longifolene in the S.cerevisiae.•Longifolene production was 36 mg/L in batch fermentation. Longifolene is a woody aroma compound that is typically extracted from plants and has strong antimicrobial activity. In this study, three potential longifolene synthase candidates from Pinus trees were tested for the de novo production of longifolene in Saccharomyces cerevisiae. Among them, the longifolene synthase from Pinus sylvestris (PsTPS) exhibited the highest catalytic efficiency, leading to the production of 1.22 mg/L longifolene in S. cerevisiae. Longifolene production in engineered yeast was optimized using multiple metabolic engineering strategies. For example, the acetyl-CoA flux in the MVA pathway was enhanced by overexpressing the genes atoB from E. coli and tHMG1, IDI1, and ERG20 from S. cerevisiae; the ethanol metabolic pathway was downregulated; the expression genes in the MVA pathway was performed using newly characterized constitutive promoters; and the DGA1 gene was overexpressed to promote the biogenesis of lipid droplets and enhance longifolene accumulation. Finally, the engineered strain produced 17.7 mg/L longifolene in a shake flask and 36.8 mg/L during fed-batch fermentation.