The embodiment of the invention discloses a fluorescent microsphere and antibody coupling method. The method comprises the following steps: (a) activating fluorescent microspheres, and resuspending toobtain a microsphere suspension; (b) adding an antibody solution into the microsphere suspension for reaction, carrying out ultrasonic treatment, and continuing the reaction; (c) after the reaction is finished, adding a confining liquid into a reaction liquid for confining, centrifuging, and collecting precipitates; and (d) putting the precipitates into a glycine solution, and carrying out ultrasonic dispersion to obtain a fluorescent microsphere and antibody coupled compound. In the coupling method, the fluorescent microspheres are activated, and sufficient contact of reactants is ensured through the ultrasonic treatment so that coupling efficiency and coupling strength of the fluorescent microspheres and the antibodies can be significantly improved, microsphere surfaces and antibody protein binding sites can be fully combined, stability of the prepared compound can be effectively improved, and an agglutination phenomenon can be avoided; in addition, sensitivity of fluorescence detection can be improved.