Disclosed are a method for marking 5-formyl cytosine and the use thereof in single base resolution sequencing. The method for marking the 5-formyl cytosine comprises the following steps of: (1) preparing a DNA or RNA sample; and (2) mixing the DNA or RNA sample with a buffer solution and a compound R1—CH2—CN to obtain a marking reaction system; and reacting the compound R1—CH2—CN therein with the 5-formyl cytosine in DNA and RNA molecules, and thereby achieving the marking of the 5-formyl cytosine; the reaction process is as in (I) below: wherein, R1 is an electron withdrawing group next to the CH2 group, preferably —CN, (II) or (III), and more preferably —CN; R is a DNA or RNA molecule connected to the 5-formyl cytosine; and the pH value of the marking reaction system is 7.5-9. On this basis, also provided in the present invention is a sequencing analysis method for the 5-formyl cytosine. The method can be implemented at a single cell level, and can achieve the sequencing of single-base resolution levels.