Ergothioneine (EGT) is a natural amino acid derived from histidine, which has many active functions such as anti-oxidation, anti-inflammation, anti-cancer and prevention of neurodegenerative diseases. Pleurotus eryngii is a delicious edible fungus with high EGT content. The purpose of this study was to explore the synthesis mechanism of EGT in P. eryngii and realize heterologous expressio of EGT in Saccharomyces cerevisiae. The EGT synthase genes in P. eryngii were discovered by bioinformatics method, and four synthase genes were obtained: PeEgt1, PeEgt2a, PeEgt2b and PeEgt2c, which were heterologously expressed in S. cerevisiae IMX581 for functional identification. Single-, double- and triple-gene heterologous expression strains were constructed, and the results showed that S. cerevisiae could synthesize EGT only by integrating PeEgt1; then integrating PeEgt2a or PeEgt2b on this basis could significantly increase EGT production, while integrating PeEgt2c did not increase EGT production. The yield of EGT of MX581-PeEgt1-2a-2b was not significantly higher than that of the double-gene engineered strains, suggesting that the low enzyme activity of PeEgt1 led to the accumulation of S-adenosylmethionine or hercynine, which might be the key rate-limiting step. The EGT synthase genes in P. eryngii were mined and successfully heterologously expressed in S. cerevisiae, and the synthesis pathway of EGT in P. eryngii was speculated. The results of this study enrich the resource pool of EGT synthetic genes, and provide ideas for the construction of engineered strains with high EGT expression.