The invention relates to a preparation method of an aggregative state phosphorescent copper nano cluster and application thereof and belongs to the technical field of nano materials. The method comprises the following step that under the room temperature, a certain amount of reducing agents and protective templates are added into a mixed solvent to be sufficiently stirred, then Cu2+ is added, even stirring is conducted for one minute and then a mixture forms into the copper nano cluster. The formed copper nano cluster has a certain phosphorescent characteristic. A biological sample solution is added into the copper nano cluster, a fluorescence resonance energy transferring phenomenon occurs between the copper nano cluster and a biological sample, and the phosphorescent characteristic of the copper nano cluster is enhanced.