Bacillus cereus (B. cereus) is a bacterial pathogen, which is more commonly found in milk and milk products and it is of particular concern to the infant formula industry. However, it can also be found in turkey, beef, rice, mashed potatoes and vegetable sprouts and causes diarrhoeal and emetic type of food poisoning. Therefore, real-time detection of B. cereus is vital for food quality monitoring. The prime intention of this paper is to pioneer the design and fabrication of a single-strand (ss) DNA biosensor without modifying the ss-probe DNA. The conducting polypyrrole modified surface is used as an immobilization matrix. The polypyrrole (PPy) film is formed on the gold electrode surface by electrochemical polymerization of pyrrole with MgCl2 as a doping electrolyte. 1μg of 20-mer ss-probe DNA specific for the B. cereus have been immobilized on the PPy film by physio-adsorption. Increase in electron transfer resistance was observed after the immobilization of the probe compared to the impedance spectra obtained for the polypyrrole modified surface. After the hybridization of the 20-mer target DNA, a further increase in impedance was noted and it is due to the addition of negative charges to the PPy/ss-DNA probe modified surface in the form of complementary DNA. Control experiments were performed to prove the specificity of the biosensor in the presence of 21-mer non complementary oligonucleotide and no unspecific bonding with the immobilized probe was observed. The performance of the DNA sensor proved to be effective in terms of selectivity, sensitivity and reproducibility of hybridization events.