Methyl donors, a class of compounds that supply methyl groups to methyl acceptors, play important roles in the function, growth, and proliferation of cells; however, the methyl donor content in cells is not sufficient to meet their normal needs. In L-tryptophan production with E. coli, the growth and acid-producing ability of E. coli cells are weak due to the presence of exogenous plasmids that inhibit the growth of E. coli, and reduce the efficiency of exogenous gene expression. Therefore, the effect of methyl donors on L-tryptophan production was investigated. Among the methyl donors tested, choline chloride showed the most significant effect in promoting fermentation, followed by methionine. The optimum addition method involved the addition of 1.5 g/L methionine to the culture medium, combined with continuous feeding with a glucose solution containing 1 g/L choline chloride. The final tryptophan titer reached 53.5 g/L; the highest biomass of bacteria reached 51.8 g/L; and the main by-product, acetic acid, was reduced to 2.23 g/L, which had a significant impact on the fermentation results.