Development of DNA probe for detection of food borne pathogen is an emerging field. Bacillus cereus strain BIS59 isolated from radiorised shrimps contains plasmid. which is 21 kb in size and is responsible for the production of non-hemolytic toxin. In this study, the detection of plasmid in B. cereus provides a basis to develop the probe for detection of B. cereus. The restriction digestion of this isolated plasmid with EcoRl enzyme revealed multiple bands. This provided a useful way to construct a plasmid library originated from the B. cereus. The studies on restriction digestion with different restriction enzymes (HindIII, NcoI, NdeIII, KpnI, BcII, BamIII, HaeIII, HpaII, XbaI and SacI) have shown insufficient fragments which were unsuitable for probe development. In order to develop the probe and to detect toxic gene, a 3 kb size fragment obtained from the B. cereus plasmid using BglII restriction enzyme. Validity of the probe was evaluated by using 43 bacterial cultures. The sensitivity of the probe was verified including standard Bacillus species and food isolates of other bacterial species by autoradiography. All the pathogenic Bacillus species were found to be hybridizing with the probe. This toxin gene based DNA probe showed high specificity to identify B. cereus. (c) 2005 Elsevier Ltd. All rights reserved.