Abstract: Provided in the present invention is an enzyme reaction solution for constructing a sequencing library and the use thereof. The enzyme reaction solution comprises an enzyme composition and a reaction buffer, wherein the enzyme composition comprises a nucleic acid endonuclease, a DNA polymerase, and a polynucleotide kinase; and the reaction buffer comprises a metal salt, a substrate, and a buffer medium aqueous solution. The present application aims to optimize the formulation of an enzyme reaction solution. The cleavage, terminal repair and addition of A to the terminal of a nucleic acid sample is achieved by a one-step reaction. In a suitable buffer system, the enzyme digestion reaction rate and the terminal repair reaction rate reach a balance. In the case where the initial amount of the sample is 100 pg to 1 µg and the processing time is the same, a sequencing library with a consistent length distribution is obtained.