The invention relates to a screening method of positive substances for a pyrolyzate trapping substance cytotoxicity test. The screening method comprises the following steps of: determining thermal cracking temperature according to the combustion state of substances participating in combustion; selecting some commonly used positive substances used for the cytotoxicity test as alternative substances of the positive substances used for the pyrolyzate trapping substance cytotoxicity test; determining whether to enter the follow-up screening step according to the boiling point, melting point, heat stability and other physicochemical properties of the alternative substances; performing the heat cracking test for the substances of which relevant physicochemical properties are failed to be inquired or the substances which have the boiling point below the combustion temperature and are not decomposed or decomposed a little during combustion; enriching the pyrolyzate; separating and analyzing through a gas chromatography-mass spectrometer; performing the heat cracking test for the substances failed to be decomposed or decomposed a little under the combustion temperature; performing MTT (Methyl Thiazolyl Tetrazolium) cytotoxicity test for the trapped pyrolyzate; obtaining the substances with cell inhibition not less than 80% to be used as the positive substances for the pyrolyzate trapping substance cytotoxicity test. By adopting the method, the positive substances for the pyrolyzate trapping substance cytotoxicity test can be screened effectively.