Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding  Escherichia coli  strain, the ergothioneine synthesis gene cluster  egtABCDE  from  Mycobacterium smegmatis  and  egt1  from  Schizosaccharomyces pombe  were introduced into  E. coli  BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of  serA T410STOP   and  thrA  resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of  serA T410STOP   and  thrA  increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1- thrA-serA  *  produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.