The invention discloses a method for preparing fluorescent microspheres immunochromatographic test paper strip and quantitative detection method. The invention takes the luminous nano-particles of dual-structure silicon dioxide compound organic dye as a marker, uses the immunochromatographic technology for preparing fluorescent microspheres immunochromatographic test paper strip, and then prepares a detection card which consists of a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper, wherein the nitrocellulose membrane is fixedly provided with a detection line and a quality control line. In the detection process, the best excitation light souce of fluorescent microspheres is used for excitation; after the emitted fluorescence passes through a filter, a CCD scanning technology or fiber-optic technology is used for collecting, accumulating or multiplicating the emitted spectra which is then converted into a numerical signal; then the measured fluorescence intensity of the detection line is multiplied by a correction coefficient, and later the corrected fluorescence intensity is substituted in a standard curve which is preset in a fluorescence analyzer; and finally, the concentration of an object to be measured in the sample can be automatically calculated and obtained by the fluorescence analyzer. The invention has high sensitivity, accurate quantization and easy operation.