The invention belongs to the technical field of luminescent materials, and particularly relates to a pure organic room temperature phosphor material and a preparation method and application thereof. The preparation method comprises the following steps that 1, carbon quantum dots with abundant elements N and O and biuret are mixed and dissolved into water or ethyl alcohol on the basis that the mass ratio of the carbon quantum dots to the biuret is 1:(40-40,000), and the mixture are stirred to be uniform to obtain a reaction mixture; 2, the reaction mixture obtained in the step 1 is filtered, a filtrate is dried and ground in sequence, and the pure organic room temperature phosphor material is obtained. According to the method, the raw materials are purely organic, low in price and environmentally friendly, the synthesis method is simple, can be implemented under normal temperature and normal pressure, and is suitable for large-scale production; the prepared pure organic room temperature phosphor material is safe, environmentally friendly, long in decay life, the decay time can reach several seconds, and the great application and popularization value is achieved on the aspects of light-emitting devices or phosphorescence materials or counterfeiting resistance or the like.