The invention discloses a two-step detection method of PCR (Polymerase Chain Reaction) of an emulsion, which belongs to the technical fields of biomedicine and clinical medicine. The method specifically comprises the steps of preparing a water-in-oil emulsion first, and then loading a PCR tube, filled with the water-in-oil emulsion, into a PCR instrument for amplification for the first step; then centrifuging an amplified product, and discarding an upper oil phase, so as to prepare an oil-in-water emulsion, loading into the PCR tube for amplification for the second step, and performing agarose gel electrophoresis detection after amplification is ended. According to the two-step detection method, the yield is further increased, and the gene detection sensitivity is increased; the two-step detection method has the characteristics of interference resistance and high specificity of the PCR of the emulsion and has the characteristic of high yield of the PCR of the emulsion in a conventional PCR method, and additionally, the processes of diethyl ether demulsification, purification and the like are not applied, so that the effects of simplicity, convenience and economy are achieved.