The invention discloses a PCR amplification detection method, which comprises the following steps: 1) constructing a PCR reaction system including: a buffer solution, a dATP solution, a dCTP solution, a dGTP solution and a dTTP solution, an upstream primer, a downstream primer and a probe, that are complementary with a detection sample, and a bis-hot-start DNA polymerase, which is formed by mixing chemical-modified hot-start Taq enzyme and nano-antibody-blocked hot-start Taq enzyme according to concentration ratio of 1:1; 2) blending the PCR reaction system with a detection sample to perform PCR amplification; 3) acquiring circulation threshold value after the PCR amplification, and according to the circulation threshold value, determining the quantity of a to-be-detected substance in the detection sample. In the invention, the nano-antibody is selected for preparing the hot-start Taq enzyme to form the bis-hot-start DNA polymerase, so that the PCR amplification detection method not only can simulate a polymerase monoclonal antibody for application in hot-start PCR, but also can functionally replace the monoclonal antibody, which is poor in stability and is greatly reduced in preparation cost.