The invention discloses a preparation method of an expandable sleeve-type micro-fluidic chip, which comprises the following steps of: S1: placing a preset object matched with a passage size into PDMSprepolymer, carrying out heating and polymerization on the PDMS, and cutting the PDMS into PDMS blocks; S2: removing the preset object, and reversing a tube seat for placing a passage, wherein the tube seat is provided with two tube orifices; S3: using an dispensing syringe subjected to chamfering polishing, and opening through holes on the PDMS blocks in a direction perpendicular to the tube seat; S4: respectively bonding both surfaces of the PDMS blocks, which are provided with the through holes, with a substrate and a top layer, wherein a sampling hole is preset on the top layer; S5: inserting a capillary glass tube with a uniform inner diameter from one tube orifice of the tube seat, inserting a capillary glass tube subjected to projection in advance from the other tube orifice of thetube seat, and completing manufacturing of a single-stage sleeve-type micro-fluidic chip; and S6: reusing the capillary glass tubes and repeating the step S5 to form a multi-stage sleeve structure. According to the invention, the operation difficulty and economic cost of manufacturing of the sleeve-type micro-fluidic chip are effectively reduced.