The invention discloses high-fluorescence nano-fluorescein organic silicon dots and a preparation method and application thereof. The high-fluorescence nano-fluorescein organic silicon dots are obtained through a reaction between fluorescein and a silylated reagent according to the molar ratio of 1:2-2:1. The prepared and synthesized high-fluorescence nano-fluorescein organic silicon dots have high fluorescence, the emitted highest fluorescence quantum yield can reach 100%, the synthetic yield is high, morphology is uniform, fluorescence stability is good, and the silicon dots have good biocompatibility and stable photofluorescence bleaching resistance and silicon nano-fluorescence optical control; and meanwhile the preparation method is easy and convenient to implement, low in cost and suitable for industrial production application, and raw material sources are wide. The prepared high-fluorescence nano-fluorescein organic silicon dots have a large red shift, show good fluorescence spectral properties depending on excitation wavelength and other advantages, and can be used for preparing a fluorescence probe based on various excellent characteristics to serve as a fluorescence marker which is applied to cell imaging and multicolor cell imaging.