5-hydroxymethylcytosine (5hmC) is a naturally existing component in mammalian genomic DNA and is regarded as the sixth DNA base. Accumulating studies have revealed the essential role of 5hmC in embryonic development, brain function and cancer research. Compared to another well-known cytosine methylation derivate, 5-methylcytosine (5mC), the detection of 5hmC is difficult for its lower lever existing in most tissues. To distinguish 5hmC from other cytosine derivates, the methods using chemical or enzymatic DNA treatment, have been applied in targeted 5hmC detection or non-targeted 5hmC enrichment. Therefore, profiling DNA hydroxymethylcytosine by sensitive, accurate and reliable method is crucial for epigenetic study. This review discusses the principles behind recently developed techniques for 5hmC quantification and mapping. By comparing the advantages and shortcomings of these methods, the general guidelines were provided on how to select appropriate methods for specific experimental contexts.