Aims: Comparison between ZEN™ double-quenched probe and SYBR GreenER™ real-time PCR assay to develop asensitive and specific assay for the direct detection and quantification of enterotoxigenic Bacillus cereus in milk.Methodology and results: Novel primers and probe were designed to target the enterotoxigenic nhe gene. Theperformance of ZEN™ double-quenched probe and SYBR GreenER™ chemistry were compared by using knownconcentrations of purified DNA. ZEN™ double-quenched probe showed a dynamic range of 3 log units and sensitivity of600 fg/reaction or 100 copies/reaction. SYBR GreenER™ chemistry had a wider quantitative dynamic range of 6 logunits with sensitivity down to 6 fg/reaction or 1 copy number/reaction. Thus, SYBR GreenER™ chemistry was 100×more sensitive with wider quantification range compared to ZEN™ probe chemistry. Similar result was also found forSYBR GreenER™ assay and ZEN™ probe chemistry in DNA extracted directly from artificially inoculated milk, with thelowest limit of detection by SYBR GreenER™ assay in the range of 6 fg/reaction or 25 copies/mL and it quantifiedBacillus cereus in milk with high relative accuracy.Conclusion: SYBR GreenER™ assay provides a fast, sensitive and specific detection and quantification ofenterotoxigenic Bacillus cereus and allowed a direct assessment and quantification of Bacillus cereus from milk foodsample.Conclusion, significance and impact of study: The study shows an efficient, specific and highly sensitive method ofdirectly assessing the enterotoxigenic Bacillus cereus from milk product, using cheaper dsDNA binding SYBRGreenER™ dye.