The invention discloses a detection method for SNP (Single Nucleotide Polymorphism) of a mixed sample based on signal spectrum difference. The detection method comprises the following steps: firstly, carrying out three rounds of independent sequencing on a wild type sample and the mixed sample by utilizing a two-nucleotide real-time synthesis sequencing technology, thus obtaining three groups of signal spectra; secondly, calculating difference between the signal spectra of the wild type sample and the mixed sample according to the signal spectra, which are obtained through three rounds of sequencing experiments, of the wild type sample and the mixed sample, and inferring an SNP site which may exist in the mixed sample by adopting an enumeration method; finally, comprehensively analyzing the probable SNP site detected through the three rounds of sequencing experiments, carrying out consistency judgment, and summarizing the position of the SNP site, a mutant nucleotide and a ratio of the mutant nucleotide if the mixed sample contains the SNP site. Compared with the prior art, the detection method disclosed by the invention has higher accuracy; a cyclic adding sequence of two nucleotides is not required to be subjected to complicated experimental design, the experiment is easier to operate, and the cost is low.