A method for specific chemical labeling of 5-formylcytosine or a 1-substituted derivative thereof, comprising the step of reacting an active methylene compound containing a side-chain active group R 1 -CH 2 -R 2 with the 5-formylcytosine or a 1-substituted derivative thereof, wherein a dehydration condensation reaction occurs between the active methylene compound containing a side-chain active group and a 5-formyl group of cytosine in the 5-formylcytosine or a 1-substituted derivative thereof, and at the same time an intramolecular reaction occurs between the side-chain active group of the active methylene compound and a 4-amino group of cytosine in the 5-formylcytosine or a 1-substituted derivative thereof to implement ring closing, as shown in the equation below: wherein, R represents hydrogen, hydrocarbyl, hydrocarbyl with -OH, -NH 2, -CHO and/or -COOH, ribosyl or deoxyribosyl, phosphate-modified ribosyl or deoxyribosyl, or structures excluding the 5-formylcytosine part of related small molecular compound or polymeric macromolecular compound formed by binding glucosidic bond in ribonucleic acid or deoxyribonucleic acid to 1-position of the 5-formylcytosine; the hydrocarbyl is C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, or C1-C30 linear or branched alkynyl, preferably C1-C10 linear or branched alkyl, C1-C10 linear or branched alkenyl or C1-C10 linear or branched alkynyl; R 1 is an electrondrawing group selected from cyano, nitro, formyl, carbonyl compound and carboxylic acid and derivatives R 2 is an electrondrawing group selected from cyano, formyl, carbonyl compound and carboxylic acid and derivatives R 3 is an unsubstituted C1-C30 linear or branched alkyl, alkenyl or alkynyl, or a C1-C30 linear or branched alkyl, alkenyl or alkynyl substituted with -OH, -NH 2, -CHO, -COOH and/or azido, or biotin; and R 1 and R 2 are independent from each other or forming a ring directly by bonding with each other or forming a ring indirectly by bonding via an atom C, N or O. wherein, R represents hydrogen, hydrocarbyl, hydrocarbyl with -OH, -NH 2, -CHO and/or -COOH, ribosyl or deoxyribosyl, phosphate-modified ribosyl or deoxyribosyl, or structures excluding the 5-formylcytosine part of related small molecular compound or polymeric macromolecular compound formed by binding glucosidic bond in ribonucleic acid or deoxyribonucleic acid to 1-position of the 5-formylcytosine; the hydrocarbyl is C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, or C1-C30 linear or branched alkynyl, preferably C1-C10 linear or branched alkyl, C1-C10 linear or branched alkenyl or C1-C10 linear or branched alkynyl; R 1 is an electrondrawing group selected from cyano, nitro, formyl, carbonyl compound and carboxylic acid and derivatives R 2 is an electrondrawing group selected from cyano, formyl, carbonyl compound and carboxylic acid and derivatives R 3 is an unsubstituted C1-C30 linear or branched alkyl, alkenyl or alkynyl, or a C1-C30 linear or branched alkyl, alkenyl or alkynyl substituted with -OH, -NH 2, -CHO, -COOH and/or azido, or biotin; and R 1 and R 2 are independent from each other or forming a ring directly by bonding with each other or forming a ring indirectly by bonding via an atom C, N or O.