A three component complex system, designated hemolysin BL (HBL), is believed to be the major diarrheal toxin of Bacillus cereus. Identification of HBL toxin by immunoassay is advantageous over PCR as it detects the expressed form of the gene, thereby differentiating pathogenic strains from nonpathogenic strains. However, most of the immunoassays, like the BCET RPLA kit, are based on the utilization of polyclonal antisera, which show cross-reactivity at times with other Bacillus species. The use of monoclonal antibodies (MAbs) binding specifically to the B. cereus HBL toxin epitopes could be advantageous. To address the problems of non-specificity of the reported detection systems and toxicity of L(1) and L(2) components during expression, we made use of recombinant chimeric rHBL protein to generate murine monoclonal antibodies. From among the L(2) MAbs stabilized, immunoblotting analyses on B. cereus strains revealed nine MAbs to be directed against the hbl D encoded L(1) protein, two to the hbl A encoded B protein, and one with the hbl C encoded L(2) protein. When tested on a large number of B. cereus standard and other related Bacillus species, there was no cross-reactivity observed among the group of MAbs. The presence of HBL component toxins among the strains recovered from food and environmental sources was evaluated by these sets of MAbs and the results compared with that of PCRs for the individual HBL toxin gene components. The HBL toxin profile characterization of the strains by Western blot using MAbs almost matched with the PCR profiles. The MAbs reported here, therefore, can be of immense help in providing the B. cereus identification/detection reliably, rapidly, and at a relatively low cost.