Here, we developed a cell-based biosensor that can assess meat freshness using the Gram-positive model bacterium Bacillus subtilis as a chassis. Using transcriptome analysis, we identified promoters that are specifically activated by volatiles released from spoiled meat. The most strongly activated promoter was PsboA, which drives expression of the genes required for the bacteriocin subtilosin. Next, we created a novel BioBrick compatible integration plasmid for B. subtilis and cloned PsboA as a BioBrick in front of the gene encoding the chromoprotein amilGFP inside this vector. We show that the newly identified promoter could efficiently drive fluorescent protein production in B. subtilis in response to spoiled meat and thus can be used as a biosensor to detect meat spoilage.