Objective To improve the responsiveness of ergosterol in electrospray ionization mode and to investigate the ergosterol derivatization through dansylation.Methods With dansyl chloride as the derivatizing agent and 4-dimethylaminopyridine (DMAP) as the catalyst, this paper carries out a response surface test to optimize derivatization conditions. The samples are separated on a C₁₈ column utilizing isocratic elution with a 0.15% formic acid-methanol solution as the mobile phase. Multiple reaction monitoring is performed using an ESI source. The ergosterol content in 10 edible fungi is assessed.Results The optimal derivatization conditions are the reaction temperature of 36 ℃, the reaction time of 25 min, and the DMAP mass concentration of 8 μg/mL. The derivatized ergosterol exhibits a broad linearity range of 25~2 000 ng/mL. The detection limit is 13.4 ng/mL, while the quantification limit is 149 ng/mL, with the sensitivity 51.94 times higher than the pre-derivatization state. Among the 10 edible fungi, shiitake mushrooms exhibit the highest ergosterol content, which is (4.21±0.083) mg/g.Conclusion This method is characterized by its ease of operation, high sensitivity, and accuracy.