Active DNA demethylation plays important roles in the epigenetic reprogramming of developmental processes. 5-formylcytosine (5fC) is produced during active demethylation of 5-methylcytosine (5mC). Here, we describe a technique called CLEVER-seq (Chemical-labeling-enabled C-to-T conversion sequencing), which detects the whole genome 5fC distribution at single-base and single-cell resolution. CLEVER-seq is suitable for the analysis of precious samples such as early embryos and laser microdissection captured samples.