In this study, the ergothioneine synthase gene Egt 1 of Pleurotus was excavated and the structure and function of its encoded protein were predicted by using bioinformatics methods. Then, an expression vector of Saccharomyces cerevisiae was constructed, and functional verification in S. cerevisiae EC 1118 was performed. The ergothioneine synthase genes PoEgt 1, PeEgt 1 and PtEgt 1 from P. ostreatus, P. eryngii and P. tuoliensis were cloned. Among these genes, the homology of nucleotide sequences was 97.03% and the homology of amino acid sequences was 97.93%. As determined by cell disruption and in vitro enzymatic reaction, the yield of ergothioneine was (2.5 ± 0.08) mg/L, which proved that the Egt 1 gene had the activity to synthesize ergothioneine.