The invention discloses a reagent for transposase fragmentation. The reagent comprises the following assemblies independently packaged: Tn5transposase, a first connector, a second connector and a transposition reaction buffer solution. Further, the reagent also comprises a forward primer set, a reverse primer set, DNA polymerase, a DNA polymerase reaction buffer solution, as well as assemblies ofa fragmentation reaction buffer solution, a stop solution and the like, wherein the forward primer set, the reverse primer set, the DNA polymerase and the DNA polymerase reaction buffer solution are independently packaged. The invention also provides a reagent kit including the reagent. The reagent and the reagent kit provided by the invention are convenient to use, low in cost, good in use flexibility and suitable for various actual requirements, and the defects that a conventional commercialization transposition system is poor in stability, single in purposes, high in cost and the like can be avoided; and particularly when the reagent and the reagent kit are used for constructing a fragmentation nucleic acid library, the process of DNA fragmentation, terminal repair and connector connection can be completed only within 10min, the library construction time can be notably shortened, and excellent sequencing quality can be obtained.