Bacillus cereus is a Gram positive rod-shape bacterium, that causes severe food poisoning. Bacillus form spores that enable it to resist the environmental stresses, such as drought, heat, pH changes. Spores can remain dormant for many years, and back to the vegetative cells when suitable conditions available for bacterial growth. Several food poisoning outbreaks in cereal products showed that B. cereus was the main causative agent. Recently, the multiplex polymerase chain reaction (m-PCR) has provide a rapid and highly sensitive method for the detection of specific pathogenic microorganisms in the aquatic environment. To date, most m-PCR assays for pathogen detection have focused on only one, two or three different types of organisms. The lack of knowledge in the development of a multiplex PCR assay for the specific detection of Bacillus cereus inspire us to spot the light on the development of the method for Bacillus cereus detection in one rapid multiplex PCR assay and the potential application of Response Surface Methodology (RSM) for the simultaneous detection of other multi-pathogen systems.