Abstract: The present invention relates to a Tryptophan-producing E. coli mutant strain CJ285 (KCCM-10534) containing single or multi mutant genes related with Tryptophan biosynthesis and production method of Tryptophan using the same. More particularly, DNA base sequences and amino acid sequences aroF, aroG, trpR, and tyrR originated from tryptophan producing E. coli mutant strain CJ285 (KCCM-10534) and related with Tryptophan biosynthesis, are disclosed, and E. coli CJ285 containing at least one of the mutant genes is cultivated directly in a glucose-containing fermentation medium, whereby L-tryptophan can be accumulated in the culture medium.