Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli–Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonoic acid, 5-deoxystrigol (5DS; 6.65 ± 1.71 μg/liter), 4-deoxyorobanchol (3.46 ± 0.28 μg/liter), and orobanchol (OB; 19.36 ± 5.20 μg/liter). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 47.3 μg/liter. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.