The invention discloses a multiplex real-time fluorescence quantitative PCR detection kit for transgenic rapeseed RF1, Oxy235 and RF3 and an application thereof. The detection kit comprises the following primers and probes: CruA-F / R, CruA-Probe, RF1-F / R, RF1-Probe, Oxy235-F / R, Oxy235-Probe, RF3-F / R and RF3-Probe. By extracting the DNA of the sample to be tested as a template, the primers and probes are added, the real-time fluorescence quantitative PCR detection is conducted, and whether the test samples contain the RF1, Oxy235 or RF3 transformant components is judged according to the markers on the probes, the Ct value and an expected amplification curve. According to the multiplex real-time fluorescence quantitative PCR detection kit, the qualitative and quantitative detection of the rapeseed internal standard gene and 3 kinds of transgenic rapeseed strains can be rapidly and accurately achieved through only one PCR reaction; the PCR detection kit has the advantages of low cost and high flux.