Most of the acute intestinal diseases are caused by food-borne pathogens. The robustness of qPCR-based fast and simple procedure for food safety detection of Mycobacterium bovis (M. bovis) DNA using EvaGreen real-time PCR for LightCycler was evaluated. Tm calling and Cp were used for analysis of PCR products. Tm calling showed better performance than Cp-based calculations for near limit of detection (LoD) positive samples. The studied qPCR M. bovis assay showed good sensitivity and excellent robustness, which allows using this assay during emergency or when this method is rarely used.