The invention discloses a pre-T carrier used for preparing eukaryon expression constructing body and a preparation method and an application thereof, relating to the technical field of T carrier preparation. The current pre-T carrier introduces Kozak sequence and XcmI box or AhdI box into current plasmid eukaryon expression carrier, utilizes XcmI enzyme digestion or AhdI enzyme digestion to form a T carrier, and utilizes the T carrier to directly clone PCR product. The preparation method of the current pre-T carrier is as follows: (1) preparing a universal insertion sequence of eukaryon expression carrier containing Kozak sequence and XcmI box; and (2) preparing the eukaryon expression pre-T carrier. The pre-T carrier in the invention exists in the form of plasmid and can be obtained continuously by the amplification of bacteria; the pre-T carrier can be used for directly cloning PCR product, thus simplifying the double enzyme digestion process on the PCR product and the carrier; the design of primer is more simple and convenient; the expression efficiency of the exogenous protein can be improved; the invention is applicable to preparing various eukaryon expression constructing bodies rapidly by the method of TA cloning.