The invention discloses a method for sequencing a gene by combing a fluorescence quantitative polymerase chain reaction (PCR). The method comprises the following steps of: performing the fluorescence quantitative PCR, performing enzymolysis, performing a sequencing reaction, purifying a sequencing product, detecting with a sequencing instrument, and the like. The invention also discloses application of the method in the preparation of a gene detection reagent kit. In the method, the fluorescence quantitative PCR mode replaces the conventional qualitative PCR and electrophoresis mode to amplify target nucleic acid and detect amplification effects, so that the detection time is shortened, the problem that PCR aerosol is polluted when a cover is opened in the electrophoresis process is solved, the false positive is reduced, and the method is extremely suitable for medical fields such as clinical detection and the like. Meanwhile, according to the quantitative results of the fluorescent quantitative PCR on target fragments, the initial concentration of the target fragments can be acquired, the concentration of PCR products can be approximately judged, subsequent gene sequencing can be directly guided, and detection sensitivity and gene sequencing efficiency are improved.