The invention discloses a paratuberculosis fluorescence PCR quick diagnosis reagent box, in particular relates to a method and a reagent box for quick detection of the paratuberculosis original bacterium-paratuberculosis mycobacteria, and belongs to the biological technology field. The invention is a group of nucleotide sequences for detecting paratuberculosis virus, and the invention is characterized in that the reagent box is the nucleotide sequences shown from the sequence table SEQ ID No.1 to the sequence table SEQ ID No.4. The invention has the advantages that: firstly, the specificity is good, the template is discriminated through a real-time fluorescence PCR technology and the specificity hybridization of a primer or a probe, the invention has very high accuracy and the false positive is low; secondly, the sensitivity is high, and a sensitive fluorescence detection system is adopted to perform real-time monitoring to the fluorescence signal; thirdly, the operation is simple, the automation degree is high, and the step of the electrophoresis detection of the product of the traditional PCR amplification is not required; fourthly, the detection is not easy to cause pollution, a closed pipe is amplified, a cover opening is not required, and the opportunity of the cross contamination and the environment opportunity is less.