Bacillus cereus is the causative agent of two types of food-borne disease،, both in raw and processed foods. Therefore, rapid and accurate detection of B. cereus is very important especially in infant formula. This study describes fabrication of a based DNA biosensor on single stranded DNA (ssDNA) of nheA virulence gene in B. cereus ATCC 14579 ( Bc 14579). ssDNA was self-assembled by thiolar binding on Au-nanoparticle modified pencil graphite electrode (PGE). The size, shape and dispersion of the G were characterized by field emission scanning electron microscope (FESEM). Detection of Bc 14579 was carried out based on an increase in the charge transfer resistance (R ct ) of the biosensor due to hybridization of the ssDNA with target DNA. Atomic force microscope (AFM) was used to confirm the hybridization of ssDNA with target DNA. The biosensor sensitivity in pure cultures of B. cereus was found to be 10 0 (CFU/mL) with a detection limit of 9.4 × 10 ? 12 mol L -1 . The biosensor could differentiate complementary DNA strand from mismatch DNA sequence and could exclusively and specifically detect the target DNA sequence of B. cereus from DNA sequence which can be found in dairy products. Moreover, the DNA biosensor exhibited high reproducibility and stability, thus it could be used as a suitable biosensor to detect B. cereus and to become a portable system for food quality control. Second part of this study was conducted to validate the performance of a fabricated DNA biosensor to detection of B. cereus in milk and infant formula. For this purpose, both culture and real-time PCR as standard methods were used. Fabricated DNA biosensor proved to be as accurate as real time PCR when quantitatively analysis culturable and non- culturable fraction of Bc 14579. The detection limit of both real-time PCR and fabricated DNA biosensor in milk and infant formula samples for detection of B. cereus ATCC 14579 ( Bc 14579) was calculated as 10 0 CFU/mL whereas for culture method it was observed to be more than 10 1 CFU/mL. In addition, three DNA extraction methods e.g silica-based column kit, boiling of bacterial pellet and cetyl trimethylammonium bromide (CTAB) were evaluated. It was shown that boiling was as equally efficient as other standard DNA extraction methods. These results suggest fabricated DNA biosensor could have a promising future outlook to be used in food quality control by microbial analysis of major food born pathogen such as B. cereus in a highly specific and sensitive way in a short time with less expertise needed to run the tests. Keywords: Electrochemical DNA biosensor, Bacillus cereus , Real time PCR, DNA extraction