The invention discloses an HPLC determination method for the gibberellin content in sugarcane leaves. The HPLC detection method comprises the following steps: (S1) adding liquid nitrogen into to-be-detected sugarcane leaves, grinding, carrying out methanol extraction and centrifugation, extracting supernate, extracting residues with methanol, extracting supernate after the centrifugation, combining the types of supernate, carrying out reduced pressure vaporization, carrying out petroleum ether extraction and decoloration, carrying out reduced pressure steaming on a low-layer water phase, adding a flow phase to dissolve, carrying out volume fixation, and filtering to obtain liquid as a sample solution for later use; and (S2) determining the gibberellin content in the sample solution by virtue of an HPLC. By virtue of the HPLC determination method, the gibberellin content in the sugarcane leaves can be determined, and the scientific basis is provided for the research of a biochemical mechanism of the gibberellin in the mediation of the stress response of pathogenic bacteria of the top rot of the sugarcanes.