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Background: L-tryptophan (L-Trp) is a nutritionally essential amino acid that the body uses to synthesize proteins, niacin (vitamin B3) and serotonin (neurotransmitters). Human and animals depend on plants and microorganisms for its supply. Remarkable increasing demand has caused the development of a wide variety of biotechnological methods for its production which work as well for other amino acids. Objectives: The present work reports on the use of Iranian sugar beet molasses as an inexpensive source of L-serine (precursor) and PLP (cofactor) for the production of L-Trp by Escherichia coli (ATCC 11303). Carbon source and buffered condition are also optimized for L-Trp production by E. coli. Materials and Methods: E. coli (ATCC 11303) was used as the microbial source of tryptophan. Batch fermentation was performed with 8L sugar beet molasses medium. The fermentation was carried out at 37 °C with an agitation speed at 250 rpm. The pH was controlled at 7 with 10 N NaOH. The cells were recovered by centrifugation. A 1g mass of E. coli cells (wet mass) was used as the biocatalyst in the reaction medium (100 cc) containing (in grams/100cc): Molasses/glucose=3.5; Indole=0.2; L-Serine=0.35; PLP=0.005; (NH4)2SO4 =0.5. The reaction medium was incubated on rotatory shaker (180 rpm) for 8 h at 37 °C. The reaction was carried out in KH2PO4-K2HPO4 buffering system (pH = 8, 0.1 M). The detection of produced L-Trp was carried out by the use of chromatography methods (HPLC and TLC), and fluorescence analysis. Results: Consequently, the potassium phosphate buffer system was considered as the suitably buffered reaction medium. The concentration of L-Trp produced in reaction medium was 0.3 mM and 0.12 mM for glucose and molasses (a combination of sugar beet and cane molasses), respectively. Conclusions: our present study shows that sugar beet molasses is a source of PLP and L-Ser; and hence, suggests the general application of the approach to produce L-Trp more economically.