Tryptophan (L-Trp) is a fundamental precursor to a number of drugs. In this research, three natural polymers included agar, agarose and alginate were investigated for immobilization of E. coli cells for L-Trp production to increase enzymatic stability. Also, the capability of beet molasses as serin (L-Ser) substitution in production reaction was investigated. According to the results, optimum conditions for L-Trp production by immobilized biocatalyst were: agar with 2 % (w/v) as support matrix, 2 g of immobilized bacterial cells as biocatalyst. The immobilized biocatalyst showed acceptable operational stability, maintaining more than 80 % of the initial activity after 5 cycles and 0.604 g/l L-Trp was produced by 2 g of immobilized cells in comparison to 0.140 g/l by 3 g of free biocatalyst. Furthermore, results showed that beet molasses can be used as a cheap source of L-Ser in the L-Trp production reaction. As a consequence, combination of immobilization and cheap substrate was successfully developed.