The invention discloses a HPLC detection method for the malic acid content of a sugarcane leaf. The method comprises the following steps: S1, adding a to-be-detected sugarcane leaf into liquid nitrogen for trituration, then adding an aqueous methanol solution with a concentration of 70 to 90%, carrying out grinding to obtain slurry, carrying out extraction at 2 to 5 DEG C, then carrying out ultrasonic extraction and centrifugation, taking a supernatant, adding the aqueous methanol solution with a concentration of 70 to 90% into residues for ultrasonic extraction, carrying out centrifugation, taking another supernatant, repeating extraction and centrifugation of the residues once to three times, combining all the supernatants, fixing the combined supernatants to a certain volume with methanol with a concentration of 70 to 90% and carrying out filtering; and S2, detecting the content of malic acid in a sample solution by using HPLC. The method can accurately detect the malic acid content of the sugarcane leaf and provides scientific and powerful detection technology for research on difference of sugarcane varieties in inductive reactance to top rot and biochemical mechanism of stress response to pathogens of malic acid-mediated top rot of sugarcane.