The invention discloses a multicolor luminous carbon nanodot as well as a preparation method and an application thereof, belonging to the field of nanomaterial science and solving the technical problems that the emitted light peak position of a carbon nanodot generates red shift and the strength is weakened with increase of wavelength of excited light and a preparation method of the carbon nanodot is high in cost, complex to operate, time-consuming and labor-consuming in the prior art. According to the preparation method, with a polycarboxylic or polyhydric organic compound or an amino acid as a carbon source and long-chained organic diamine as a surface passivation modifier, the multicolor luminous carbon nanodot is prepared by the steps of low-temperature pyrolysis, precipitation washing, dialysis separation and freeze drying. The preparation method of the multicolor luminous carbon nanodot is simple, low in cost and can be conveniently put into large-scale production; the prepared carbon nanodot is at a solid state, is convenient to store, has high fluorescence quantum efficiency and good biocompatibility, can respectively emit strong green light, orange light and red light under the excitation of blue light, green light and yellow light, and can be used as a bioluminescence probe and an optical imaging marker.