A method of measuring the content of mannan in a yeast culture product is disclosed. The method includes main steps of (1) pretreating the yeast culture product to break walls of yeast cells in the yeast culture product; (2) subjecting the pretreated sample to hydrolysis under acidic conditions; (3) adjusting the pH value of a solution obtained after the sample is subjected to hydrolysis to be neutral, performing derivatization with a corresponding derivatization agent under certain conditions, and subjecting a mannan standard substance solution to derivatization under same conditions; (4) subjecting the derivatized sample and the derivatized standard substance solution to analysis and detection by reversed-phase high-performance liquid chromatography, with mannan being adopted as a standard substance solution; and (5) calculating the mass of the mannan in the sample according to obtained chromatographic data, and then converting the mass into the content of the mannan. The content of the mannan in the yeast culture product can be accurately measured by the method, interferences are few and specificity is high.