The invention discloses a carbohydrate antigen CA125 quantitative determination kit. The kit comprises a calibration substance, a quality control substance, a resisting reagent, a magnetic particle reagent and a luminescence substrate. The invention also discloses a making method of the kit, and a method for detecting a carbohydrate antigen CA125 by using the kit. The resisting reagent is prepared by adopting a fluorescein isothiocyanate labeled CA125 coated antibody and an alkaline phosphatase labeled CA125 labeled antibody, and the magnetic particle reagent is prepared through coupling an anti-fluorescein isothiocyanate antibody with a carboxyl magnetic sphere, so uniform mixing and separation are easy in an immunization reaction, and the reaction speed is greatly improved; and a novel chemiluminescence substrate APLS is adopted as the substrate, so the sensitivity and the specificity of the kit are improved. The determination kit has the advantages of reliable performances, high sensitivity and wide linear range, and can be used together with a semi-automatic and fully-automatic apparatus.