A determination method of an acrolein-DNA adduct in saliva is a determination method of alpha-Acr-dG and gamma-Acr-dG in DNA, and concretely comprises the following steps: collecting saliva through adopting an OG-500 saliva collecting tube, carrying out DNA extraction on the saliva, carrying out enzyme hydrolysis on the obtained DNA solution, and sequentially adding a stable isotope internal standard substance and DNA hydrolase; and carrying out Strata-X column solid phase extraction on the obtained hydrolysate, collecting the obtained eluate, blowing the eluate with nitrogen at room temperature until the eluate is dry, re-dissolving the dried eluate in 100[mu]L of a 30% methanol-water solution, introducing the obtained new solution to an LC-MS/MS system, and accurately detecting the content levels of the alpha-Acr-dG and gamma-Acr-dG. The stable isotope is adopted as an internal standard quantitative analysis substance in order to reduce the error caused by a pretreatment process, and tandem mass spectrometry well improves the selectivity, the accuracy and the sensitivity of the method. The method well improves the separation degree of target objects through selecting a chromatographic column and optimizing the eluting conditions.