The invention discloses a urine preservation reagent and an application thereof. The urine preservation reagent comprises a nucleic acid buffer, a metal ion chelating agent, an antiseptic, a cell fixing agent, and a formaldehyde quenching agent; a nucleic acid buffer is Tris-HCl, a working pH value is 7-8, the metal ion chelating agent is EDTA, the antiseptic is citric acid and/or sodium citrate,the cell fixing agent is selected from at least one of paraformaldehyde, diazolidinyl urea, imidazolidinyl urea, and diethyl urea; and the formaldehyde quenching agent is selected from at least one ofglycine, lysine, ethene diamine and arginine. The urine preservation reagent can effectively preserve free DNA in urine, degradation can be avoided, and the urine preservation reagent establishes a base for subsequently detecting the free DNA; fragmentation of cells in the urine is prevented, a genome DNA background is reduced, and free DNA detection result accuracy is guaranteed. The urine preservation reagent is the novel reagent for preserving a urine sample, and provides the guarantee for clinical detection of the urine free DNA.