The invention relates to the technical field of biological and medical detection, and particularly relates to a time-resolved fluorescence immunoassay kit for comprehensively detecting uterine cancer tumor markers SCC (Squamous Cell Carcinoma), CEA (Carcinoma Embryonic Antigen), CA125 (Carcinoma Antigen 125), and beta-HCG (Human Chorionic Gonadotropin) simultaneously in a same reaction system, and further relates to application of the kit in comprehensive detection of the uterine cancer tumor markers SCC, CEA, CA125, and beta-HCG simultaneously. The kit comprises a first monoclonal antibody mixed antibody coated microplate for respectively resisting SCC, CEA, CA125, and beta-HCG, a second monoclonal antibody mixed antibody of Eu<3+> labeled anti-SCC, Sm<3+> labeled anti-CEA, Tb<3+> labeled anti-CA125 and Dy<3+> labeled anti-beta-HCG, an analytical buffer solution, a common fluorescence reinforcing agent, a washing liquid and a quality control product. The kit related by the invention can be used in clinical auxiliary diagnosis, curative effect observation, and prognosis judgment of the uterine cancer, has a great significance on the treatment and prevention of the uterine cancer, and can be used for detecting the four tumor markers simultaneously, thereby simplifying detection steps, and improving the specificity and the sensitivity of the comprehensive analysis of detection data.