The invention relates to the technical field of bioinformatics, particularly to a whole-genome methylation non-bisulfite sequencing library, a construction and applications thereof, wherein the sequencing library comprises a TET enzyme reaction solution, the TET enzyme reaction solution comprises the following independently packaged components: a TET enzyme oxidation buffer solution, and the TET enzyme oxidation buffer solution comprises, by micromole, (20-167)*10<3> parts of HEPES or Tris-Cl, (100-333)*10<3> parts of NaCl, 3.3*10<3> parts of alpha-KG or 2-oxoglutarate, 6.67*10<3> parts of ascorbic acid and 4*10<3> parts of adenosine triphosphate. According to the invention, by combining the kit, Fe(NH4)2(SO4)2 and TET enzyme, 5mc can be oxidized into 5cac, the 5cac is reduced into dihydrouracil under the action of a reducing agent, and T is identified through PCR sequencing, so that the the DNA methylation C-to-T conversion under the non-bisulfite condition is achieved, the defects ofbase imbalance and low sequencing data use rate of the existing methylation sequencing library constructed based on the bisulfite conversion are solved; and the formula further has effects of simplecomponents, extremely low TET enzyme use amount and significant cost reducing.