Abstract: Objective To investigate the diagnostic value of human cytomegalovirus(HCMV) DNA detection results in children's blood, urine and mother's breast milk in HCMV infection. Methods A total of 328 children with suspected HCMV infection who were admitted to the hospital from 2017 to 2018 were enrolled. Blood, urine and corresponding breast milk samples were collected for real-time fluorescent quantitative PCR to detect the HCMV-DNA load. Serum HCMV-IgM was detected by chemiluminescence method. The positive rates of different methods in different samples were compared. For positive children, the distribution of different clinical symptoms was analyzed. Results The total positive rate of 328 children was 71.04%, and the positive rates of male and female children were 71.02% and 69.74%, respectively, with no statistically significant(χ²=0.065, P=0.799). The positive rates in blood, urine and milk were 49.09%, 70.43% and 58.23%, respectively, and the positive rate of urine was the highest, with statistically significant(χ²=31.147, P<0.001). The positive rate of milk(72.41%) in the neonatal group was the highest. The positive rate of urine was highest in children between 28 days and 1 year old and 1 year old. The positive rate was statistically different among all age groups(all P<0.05). The positive rates of blood HCMV-DNA and serum HCMV-IgM were 49.09%(161/328) and 16.16%(53/328), respectively. The positive detection rate of the combined detection of the two methods was 49.69%. There was no statistically difference significant between combined detection and single blood DNA detection method(P>0.05). The main clinical symptoms were jaundice and diarrhea in children under one year old, and liver damage and pneumonia in children over one year old. Conclusion The detection of urine HCMV-DNA has a better clinical value in the diagnosis of HCMV. At the same time, urine is convenient to take, and urine HCMV-DNA detection should be promoted in children with small age.