Abstract: OBJECTIVE To establish the method for determining ergosterol peroxide in marine fungi Aspergillus sp. by HPLC-ELSD. METHODS Marine fungi Aspergillus sp. was cultured by artificial fermentation, fermentation broth was extracted by ethyl acetate, and determined by HPLC-ELSD. C₁₈ column was used with the mobile phase of methanol and water gradient elution, and the determination condition of evaporative light detector was drift tube temperature of 75.0℃, atomization temperature of 30.0℃, carrier gas pressure of 40 psi, the gain was 40. RESULTS Pair value of sample quality and peak area had a good numerical linear relationship in the ergosterol peroxide concentration range of 2.5-40 μg·mL^-1, the average recovery was 100.1%, the RSD value of precision test was 1.9%, the RSD value of repeatability test was 1.61%, the RSD value of stability test was 1.46%. CONCLUSION The method has a good separation, quick analysis speed and accurate result, it is suitable for the determination of marine fungus Aspergillus sp. of peroxy ergosterol content.