Cell-free DNA (cfDNA)-based biomarkers such as mutation and methylation offer promising noninvasive strategies for disease diagnosis and prognosis. However, besides high-throughput sequencing, there has been no alternative approach to date to detect the epigenetic marks, such as 5-hydroxymethylcytosine (5hmC), in cfDNA. Here, we described a MnO2 oxidation and hydrazine-s-triazine reagent (i-Pr2N) labeling based method named 5hmC-MIQuant that achieved ultrasensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of 5hmC in low-input DNA samples. This strategy improved the detection sensitivity of 5hmC by 178 times, and the limit of detection was as low as 14 amol. With simple preparation steps, 5hmC-MIQuant could quantify the 5hmC level in as little as 340 pg genomic DNA (equivalent to 57 copies of diploid genome). cfDNA samples from human plasma were successfully analyzed using 5hmC-MIQuant. This method is promising for the identification of 5hmC function in precious samples and the 5hmC-based noninvasive disease diagnosis.