Food contains several microorganisms that may cause illnesses and food poisoning in humans. Small numbers of microorganism contamination could result in rapid spoilage of food. The Centres for Disease Control and Prevention (CDC), USA estimates that 76 million people are affected by foodborne illnesses each year in the USA. Salmonella infections alone account for one billion dollars yearly in direct and indirect medical costs and more than 5,000 deaths. In Sudan, diarrhoeal disease was reported as the second major disease during the years from 2003 to 2007 (Annual health statistical report of the Federal Ministry of Health, Sudan). We aimed to develop a rapid molecular procedure for the detection of Escherichia coli, Shigella dysentery, and salmonella Typhiin food so as to minimize the public health hazard of food contamination. We used the Multiplex PCR method as rapid methods were tested for identification of Enterobacteriaceae species Escherichia coli as an indicator organism for food contamination and two strains of Enterobacteriaceae that causes food borne illness (namely Shigella dysentery and salmonella Typhi). The Multiplex PCR was performed to detect E. coli using Mdh primer pair, Salmonella Typhi using IpaB primer pair, and Shigella dysentery using IpaH1 primer pair. The sensitivity to detect E. coli, Salmonella Typhi, and Shigella dysentery in contaminated food in the concentration of the infective and the over infective doses were 100%, 96.3%, and 88.9% respectively for the three bacteria strains. There was no significant difference in the detection of the bacteria after incubation for 8 hours, 24 hours, or even without incubation period. There were no differences in the result of the samples that were contaminated artificially in laboratory and those obtained from the market. The Multiplex PCR method for identification of E. coli, Salmonella Typhi and Shigella dysentery was developed as a model for detection and risk assessment of the three bacteria in one program, and it is suitable for routine analysis.