The invention discloses a carbon nano-dot, and a preparation method and an application thereof, and solves a problem that the application of present nano-dots is restricted because of high preparation cost and easy fluorescent quenching appearance of an aggregate state. According to the invention, the carbon nano-dot having a high fluorescence quantum efficiency is prepared through adopting a polycarboxyl or polyhydroxy contained organic compound, or an amino acid as a raw material, and urea as a surface passivation modification agent, and through a microwave process, and a carbon nano-dot fluorescent ink is prepared through using the carbon nano-dot. The preparation method disclosed in the invention has the advantages of simplicity, low cost and convenient large-scale production; the fluorescent quenching of the prepared carbon nano-dot on the surface of a biological product does not appear, and the highest fluorescence quantum efficiency is 42%; and the prepared carbon nano-dot fluorescent ink is nontoxic, does not generate a precipitate after long-time dispose, and can be applied to the biological imaging field, the biological product identification field, the information storage field, the information encryption field, the false proof field, the illumination display field, the photovoltaic device field and the like.