Nisin is a lantibiotic, an antibacterial peptide produced by certain <i>Lactococcus lactis</i> strains that kills or inhibits the growth of other bacteria. Nisin is widely used as a food preservative, and its long-time use suggests that it can be generally regarded as safe. We have developed a method for determining the amount of nisin in food samples that is based on luminescent biosensor bacteria. Bacterial luciferase operon <i>luxABCDE</i> was inserted into plasmid pNZ8048, and the construct was transformed by electroporation into <i>Lc. lactis</i> strain NZ9800, whose ability to produce nisin has been erased by deletion of the gene <i>nisA</i>. The operon <i>luxABCDE </i>has been modified to be functional in gram-positive bacteria to confer a bioluminescent phenotype without the requirement of adding an exogenous substrate. In the plasmid pNZ8048, the operon was placed under control of the nisin-inducible <i>nisA</i> promoter. The chromosomal <i>nisRK</i> genes of <i>Lc. lactis</i> NZ9800 allow it to sense nisin in the environment and relay this signal via signal transduction proteins NisK and NisR to initiate transcription from <i>nisA</i> promoter. In the case of our sensor bacteria, this leads to production of luciferase and, thus, luminescence that can be directly measured from living bacteria. Luminescence can be detected as early as within minutes of induction. The nisin assay described here provides a detection limit in the sub-picogram level per ml, and a linear area between 1 - 1000 pg/ml. The sensitivity of this assay exceeds the performance of all previously published methods.