The invention relates to a kit and a method for quantitatively detecting urine microalbumin through time-resolved fluorescence. The kit comprises a time-resolved fluorescence test paper card, sample diluent and a DS card containing a calibration curve, wherein the time-resolved fluorescence test paper card comprises a detection test paper strip which consists of a bottom liner, and a sample pad, a nitrocellulose membrane and absorbent paper which are pasted on the bottom liner; the nitrocellulose membrane is successively provided with a microsphere line, a detection line and a quality control line; the microsphere line consists of time-resolved fluorescence microspheres marked with human seroalbumin monoclonal antibodies; the detection line is coated with human seroalbumin recombinant antigens; the quality control line is coated with goat anti-mouse IgG. The kit provided by the invention can accurately and quantitatively detect the content of microalbumin in human urine; markers are used for connecting the microspheres with antibodies through covalent bonds; and the marked products are stable. The kit has the characteristics of wide detection range (2 to 300mg/ml), high sensitivity (the detection limit is 2300mg/ml), high accuracy, fast and easy detection and the like.