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To determine the vitamin B12 content in fermented foods using Riboswitch gene based selective method. Riboswitch are organized metabolite binding domains which reside within the non-coding locales of mRNAs, where they tie metabolites and serve as precision sensors for their targets corresponding, and control gene expression. A Propionibacterium freudenreichii was cloned in p519NGFP vector in Escherichia coli BL21. Based on the Adenosylcobalamin concentration, it correlates the expression using green fluorescence protein. The conformational change of secondary structure of mRNA leads to bind directly Adenosylcobalamin and inhibits the expression. After various experiments, results obtained from standard curve from 10 to 1000 ng/mL of cyanocobalamin. Outcome of the study reveals that recovery percentage is 94.5% compared with microbiological assay, HPLC but Ca. 26% lower than in Riboswitch sensor method.