Objective  To prepare an urine internal quality control substance for fluorescence PCR detection of cytomegalovirus DNA and observe the stability. Methods  Urine samples with positive CMV-DNA were collected, filtered, and preserved at -80℃ to observe its stability. The effect of preservation conditions of -80 ℃ and -20 ℃ on the stability of quality control substance was compared. Results  Among 120 tests, first 20 tests adopted the instance method and the results were under control. Levey-Jennings quality chart was plotted after 20 tests and the results were all under control according to Westgard rules. The preservation conditions of -80 ℃ and -20 ℃ did not affect the stability of prepared internal quality control substance. Conclusion  The urine internal quality control substance prepared by simple method is stable and can be used as internal quality control substance for fluorescence PCR detection of cytomegalovirus DNA.