The invention discloses a DNA (deoxyribonucleic acid) library and a method for constructing a transcription activator like effector nuclease plasmid. A transcription activator like effector connection unit library comprises 16 groups*n double-basic group identification modules and 4 groups*m single-basic group identification modules, wherein two tail ends of each double-basic group identification module or single-basic group identification module are both formed into a first cohesive tail end and a second cohesive tail end by being subjected to enzyme digestion through a second class restriction enzyme; n expresses the quantity of each group of double-basic group identification modules, and m expresses the quantity of each group of single-basic group identification modules. The DNA library comprises plasmids, wherein the plasmids are the same as the connection units of the Tale connection unit library in quantity, each plasmid comprises a connection unit with basic group sequences mutually different, the connecting positions of the two tail ends of the connection unit and an original carrier are provided with BsaI enzyme digestion sites. The DNA library disclosed by the invention is used for constructing the transcription activator like effector nuclease plasmid, can realize that 19-26 identification modules are connected for one step by identifying the enzyme digestion of the modules and connecting in same reaction, and has the advantages of high speed, high efficiency, easiness for operation, easiness for material preservation and low cost.