Abstract: 5-Hydroxymethylcytosine (5hmC), which was formed from the enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins family, played an important role in gene transcription and expression regulation as a new epigenetic modified in mammal animal. A method for global DNA hydroxymethylation in blood sample of rats using hydrophilic interaction liquid chromatography-tandem mass spectrometry(LC-MS/MS) was developed. Genomic DNA from blood samples was extracted by genomic DNA isolation kit, and then was hydrolyzed with formic acid at 140 ℃. The LC separation was conducted on Waters HILIC column by gradient elution with acetonitrile 7 mmol/L ammonium formate as mobile phase, and the analysis were performed by tandem MS with positive ion electrospray ionization (ESI⁺) in multiple reaction monitoring (MRM) mode. The results show that the calibration curve with a good linearity in the concentration range of 0.1-50 μg/L is established for 5hmC, and the correlation coefficients are higher than 0.999. The limit of detection (LOD, S/N=3) and the limit of quantification (LOQ，S/N=10) for 5hmC are 0.050 and 0.100 μg/L, respectively. The relative standard deviations (RSD) of the intra-day and inter-day precision are 3.42% and 4.12%, respectively. The recovery of the spiked standards varies from 92.51% to 102.19%. The method is applied to analyze the hydroxymethylation level of genomic DNA from the blood samples of rats, and a average 5hmC degree of 0.48% is acquired. The method is available for the determination of global DNA hydroxymethylation in blood for its simplicity, speediness, and precision.