The invention provides an azo linkage unit based fluorescence labeled nucleotide and applications thereof. The structural formula of the nucleotide is shown in a formula VI in the specification, wherein fluorescein is selected from one of BODIPY, rhodamine, coumarin, xanthene, cyanin, pyrene, phthalocyanine, Alexa, Squaring dye, a combination producing energy transfer dyes and derivatives thereof; R1, R2, R3 R4 and R6 are various substituent groups, R5 is a substituent group except -C2H5, and R1, R2, R3, R4, R5 and R6 are not H simultaneously; and n is an integer between 0 and 10. Compared with the prior art, a kind of new azo linkage unit based reversible terminals is synthesized according to the invention; the kind of reversible terminals can realize high-efficiency shearing under moderate conditions, so that the kind of reversible terminals can be used for DNA sequencing; and raw materials required by synthesis are simple and easy to obtain, and reactions in the synthetic process are all conventional chemical reactions, therefore, the nucleotide can be subjected to large-scale promotion and application.