Aiming at Illumina high-throughput sequencing platform, this kit provides a convenient and universal DNA library construction scheme in one tube. It combines end repair and end “A” tailing into one step, greatly shortening the time of library construction and reducing the error caused by tedious steps. The end repair, adapter ligation, amplification and purification of the fragmented double-stranded DNA can be performed within about 2 hours. Complete library quantification can be performed by dsDNA fluorescent dye method (e.g., Thermo Qubit Flex Fluorometer) or absolute quantification PCR after diluting the library to an appropriate concentration.