The invention relates to the technical field of biological and medical detection, in particular relates to a kit capable of simultaneously and comprehensively detecting ovarian cancer tumor markers HE4 and CA125 in a same reaction system, and also relates to application of the kit. The kit comprises a porous plate coated with a first monoclonal antibody mixture which resists the HE4 and CA125 respectively, an Sm<3+> labeled anti-HE4 and Eu<3+> labeled anti-CA125 second monoclonal antibody mixture, an analytical buffer solution, a fluorescence enhancer, a cleaning solution and a quality control material. The kit provided according to the invention can be used for clinical auxiliary diagnosis, observation of curative effect and prognosis of the ovarian cancer, has significance for treatment and prevention of the ovarian cancer tumor, and can be used for detecting the two tumor markers simultaneously, thus simplifying the detection steps, and improving the specificity and sensitivity of comprehensive analysis of detected data.