The invention relates to a method for separating and determining pyrazine substances and pyridine substances in saliva, and belongs to the technical field of analytical chemistry. A saliva sample is collected through absorbent cotton, is centrifuged and undergoes solid phase extraction, and the pyrazine substances and pyridine substances in saliva are determined through gas chromatography-mass spectrometry. The method comprises the following steps: 1, collecting the saliva sample by using absorbent cotton, placing the absorbent cotton in a syringe needle cylinder after collection ends, pushing a syringe push rod to carry out extrusion in order to obtain a saliva sample, carrying out high speed centrifuge, and taking the obtained supernatant; 2, adding an internal standard substance to the saliva sample, carrying out solid extraction column enrichment, and eluting the obtained material with a solvent; and 3, concentrating the obtained eluate, re-dissolving the obtained concentrate, allowing the re-dissolved concentrate to go through a millipore filter membrane, determining the filtered solution through gas chromatography-mass spectrometry, making a standard curve, and carrying out internal standard method quantification. The method can be effectively used to determine pyrazine substances and pyridine substances in saliva, and has the advantages of feasibility, low detection limit, good precision, high accuracy and reliability, and promotion application values.