The invention discloses a fluorescence quantitative PCR detection kit of hepatitis B virus and an application thereof. The kit is composed of the following independent components: DNA extraction solution I, DNA extraction solution II, DNA extraction solution III, DNA extraction solution IV, positive control interior label, PCR reaction liquid, probe HBV-SP, enzyme mixed liquor containing heat resistant DNA polyase and uracil DNA glycosylase, quantitative hepatitis B virus reference material, hepatitis B virus positive control serum and hepatitis B virus negative control serum, wherein DNA extraction solution I contains 0.2-1.0% of lauryl sodium sulphate (mass/volume), 1.0-4.0% of Triton (volume/volume) and 0.2-1.0mol/L of guanidinium isothiocyanate; DNA extraction solution II contains 100-300mmol/L of 4-HEPES, 100-300mmol/L of sodium chloride with pH of 6.5+/-0.2 and 100-400 mu g/ml of magnetic beads; DNA extraction solution III contains 0.1-1.0% of Triton (volume/volume) and 100-300mmol/L of sodium chloride; DNA extraction solution IV contains mineral oil. The fluorescence quantitative PCR detection kit of hepatitis B virus of the invention can be used for detecting the HBV-DNA concentration in samples of serum, blood plasma or latex and the like.