The invention discloses a two-step enzymatic determination method for 
cholesterol ester in serum, which belongs to the field of methods for testing materials through color change of test reaction results by utilizing visible light. The technical scheme of the invention is as follows: a 
reagent II only contains effective components of 
hydrolase of the 
cholesterol ester, and bi-color original substances and other effective components are contained in a 
reagent I; and the determination method is as follows: firstly carrying out warm bath on the serum and the 
reagent I for 3-5min at 37 DEG C, leading 
free cholesterol in the serum to be reacted with the reagent I to generate quinonimine, adding the reagent II, then carrying out the warm bath for 4-7min at 37 DEG C, generating the 
cholesterol after 
hydrolysis of the cholesterol ester, and carrying out reaction for generating the red quinonimine. An instrument is used for detecting at the 
wavelength of 500nm-520nm, the quinonimine generated by the reaction of the reagent I is taken as blank, and the quinonimine generated by the reaction of the reagent II can be used for determining the content of the cholesterol ester. The method can not be affected by the endogenous 
free cholesterol during the detection, and the using method and the range are the same with the original two-step enzymatic method, thereby being the cholesterol ester detection method with quickness and higher accuracy.