Aims: To investigate the effects of pretreated-beet molasses on Escherichia coli fermentation using benzaldehyde lyase (BAL) production by recombinant E. coli BL21(DE3)pLySs process as the model system. Methods and Results: The effect of the initial pretreated (hydrolysed) beet molasses concentration was investigated at 16, 24, 30 and 56 g l-1 at a dissolved oxygen condition of 40% air saturation cascade to airflow, at N = 625 min-1 and pH(C) = 7 center dot 2 controlled-pH operation conditions. The highest cell concentration and BAL activity were obtained as C-X = 5 center dot 3 g l-1 and A = 1617 U cm-3, respectively, in the medium containing 30 g l-1 pretreated beet molasses consisting of 7 center dot 5 g l-1 glucose and 7 center dot 5 g l-1 fructose. Production with and without IPTG (isopropyl-beta-d-thiogalactopyranoside) induction using the medium containing 30 g l-1 of pretreated beet molasses yielded the same amount of BAL production, where the overall cell yield on the substrate was 0 center dot 37 g g-1, and the highest oxygen transfer coefficient was K(L)a = 0 center dot 048 s-1. Conclusions: Pretreated beet molasses was used in the fermentation with E. coli for the first time and it yielded higher cell and BAL production compared with the glucose-based medium. Significance and Impact of the Study: Pretreated beet molasses was found to be a good carbon source for E. coli fermentation. Furthermore, IPTG addition was not required to induce recombinant protein production as galactose, one of the monomers of trisaccharide raffinose present in the beet molasses (1 center dot 2%), induced the lac promoter.