A specific method to identify nisin-producing strains was developed based on Nisin-Controlled gene Expression (NICE) vector pSec:Nuc. The plasmid pSec: Nuc was transformed into non-nisin-producing strain Lactococcus lactis NZ9000, a host commonly used for the NICE system. The generating strain L. lactis NZ9000/pSec:Nuc could sense extracellular inducer nisin and efficiently secrete a reporter protein Nuc, the staphylococcal nuclease (Nuc) into the medium. Instead of using purified nisin, the culture supernatants of nisin-producing strains were also used as inducers. Therefore, the NICE system could be used to identify nisin-producing strains. With this principle, 4 among 56 lactococci strains isolated from raw milk were identified as nisin producers. The results were further confirmed by polymerase chain reaction amplification with their genomic DNA as templates, and nucleotide sequencing revealed that three of them produced nisin A, and the others produced nisin Z. Those results made it possible to isolate and identify nisin-producing strains specifically and rapidly using NICE system.