The present disclosure relates to the field of mass spectrometry, and particularly provides a method for determining a chemical structure of a lipid and an ion mobility-tandem mass spectrometer. The method for determining a chemical structure of a lipid includes: an ionization step of ionizing a sample to obtain sample ions; an ion mobility-based separation step of separating target lipid ions from the sample ions based on ion mobility; a first dissociation step of dissociating the target lipid ions with dissociation energy adjusted to break a first chemical bond of the target lipid ions; a mass-based selection step of selecting the target lipid ions, whose first chemical bond is broken, based on a mass number to obtain fragment ions; a second dissociation step of dissociating the fragment ions to at least break a second chemical bond of the fragment ions which has bond energy higher than the first chemical bond, to obtain diagnostic ions; and a mass analysis step of performing a mass analysis on the diagnostic ions.