The invention discloses a four-color fluorescence labeling reversible terminal and a use thereof in DNA (Deoxyribonucleic Acid) sequencing. The structural formula of the reversible terminal is as shown in a formula (I) in the specification, wherein R1 is triphosphate; R2 is H or OH; the basic group is U, C, A, G or derivatives thereof; the connecting unit is a bifunctional compound which is breakable under a mild condition; the fluorescence group is one selected from a combination of BODIPY, fluorescein, rhodamine, coumarin, xanthene, cyanin, pyrene, phthalocyanine, alexa, squarene dye and an energy transferring dye, and derivatives thereof. The reversible terminal provided by the invention can be used for DNA single-molecule sequencing; simultaneously, raw materials required by the synthesis of the reversible terminal provided by the invention are simple and easy to get and the synthesis process of the reversible terminal is completely involved with conventional chemical reactions, so that the four-color fluorescence labeling reversible terminal can be popularized and utilized to a large scale; biological evaluation results indicate that the reversible terminal is capable of completely meeting the biochemical reaction requirements of high-flux sequencing and has a good practical prospect.