Blakeslea trispora is widely used as an industrial microorganism for β-carotene production due to its rapid growth and high carotene yield per unit biomass. Carotenoids synthesized by B. trispora are extensively applied in food and pharmaceutical industries because they meet food safety standards. Compared with plant extraction, microbial fermentation offers advantages such as shorter production cycles and higher economic efficiency. To elucidate the carotenoid biosynthetic pathway in B. trispora and facilitate the construction of heterologous carotenoid-producing strains, key genes involved in carotenoid biosynthesis were cloned and subjected to phylogenetic analysis. Functional characterization of the encoded enzymes was performed using a functional complementation strategy. Recombinant Escherichia coli strains capable of producing β-carotene were successfully constructed, including E. coli-pAC-car G (1.90 mg.g-1 DCW), E. coli-pAC-car B (0.53 mg.g-1 DCW), E. coli-pAC-car RA (0.27 mg.g-1 DCW), and E. coli-pAC-car G-car B-car RA (0.33 mg.g-1 DCW). Functional analysis confirmed that car G encodes geranylgeranyl pyrophosphate synthase, car B encodes phytoene dehydrogenase, and car RA encodes a bifunctional enzyme with phytoene synthase and lycopene cyclase activities. These findings clarify the roles of key carotenoid biosynthetic genes in B. trispora and provide a foundation for elucidating its complete carotenoid metabolic pathway and for developing engineered strains for carotenoid production.