The invention discloses an HPLC detection method of the content of salicylic acid in sugarcane leaves. The method comprises the following steps: S1, adding the sugarcane leaves to be detected into liquid nitrogen, grinding the leaves, carrying out methanol extraction, centrifuging the obtained extract, taking obtained supernatant, carrying out methanol extraction on obtained residues, centrifuging the residue extract, taking obtained supernatant, mixing the supernatants, carrying out reduced pressure evaporation, carrying out extraction decolorizing, carrying out reduced pressure evaporation on obtained lower layer water phase, adding a mobile phase to dissolve the evaporated water phase, allowing the volume of the obtained solution to reach a constant value, and filtering the solution to obtain a sample solution for later use; and 2, detecting the content of salicylic acid in the sample solution through adopting HPLC. The method allows the content of salicylic acid in sugarcane leaves to be accurately detected, and provides a scientific and powerful detection technology for the top rot resistance difference research of sugarcane varieties and the stress response biochemical mechanism of salicylic acid mediated sugarcane top rot pathogens.