The invention discloses a composite nano-particle for three-dimensional fluorescence imaging of a blood vessel network, and a preparation method thereof and an application thereof. The composite nano-particle includes 1-30 wt% of a fluorescent material used as a core and 70-99 wt% of an amino-modified polymer used as a shell layer. The fluorescent material has the advantages of controllable dimensions, good water dispersibility, high light stability, and simple and easy preparation process. The prepared composite nano-particle has the advantages of high fluorescence brightness, uniformity in dyeing, low interference of photobleaching and background fluorescence, low toxicity and low cost when used for the blood vessel imaging, can accurately mark the fine structure of the blood vessel network, and has potential practical values in in-vivo imaging, medical diagnosis, disease therapy and other important fields.