Heterologous expression of natural product biosynthetic pathways is of rising interest to fungal bioactive compound discovery and engineering. However, most eukaryotic genes are regulated by individual regulatory elements and transcribed as monocistronic mRNA. Therefore, regardless of plasmid transformation or chromosomal integration of DNA, reconstruction and expression of biosynthetic gene clusters in the eukaryotic recipient require improved assembling pipeline of vectors and coordinated control of expression of multiple genes. Moreover, valuable medicine biosynthesis based on heterologous expression remains an ongoing challenge owing to the strict metabolic regulation in the chosen host. To circumvent these obstacles, a specialized vector was designed, for flexible but modularized construction of multiple genes. In the scheme, TEV protease and its recognition site combined with a previously documented P2A-mediated expression system, allow expression of eukaryotic genes in a “polycistronic” mRNA by co-translational and post-translational cleavages. As another highlight, the split fluorescent protein was introduced for visualizing and rapid screening of fungal transformants. On the basis of this new tool, the austinoid biosynthetic pathway has been rewired successfully in A. nidulans. Also, it represents the first-time producing psilocybin heterologously using fermenting condition, from which the high titer 15.4 mg/g (110 mg/L) was achieved. Subsequently, the de-branching strategy was beneficial for the yield of psilocybin, which even lifted to 57 mg/g in the yeast cell-based factory. Meanwhile, the Trp auxotrophic strain has been endowed with the capability to synthesize isotope-labeled psilocybin in an atom-economic manner.