The present invention relates to an autofocusing method for a two-photon photopolymerization nanofabrication system, which comprises the following: a laser source outputting ultrashort pulse laser beam capable of causing Twophoton Absorption Phenomenon in an autofocusing system for a two-photon photopolymerization nanofabrication system using fluorescence of pre-two-photon absorption region; a Galvano shutter converting a laser beam route extended; a mirror converting the route by reflecting laser beam outputted by the laser source; an object lens for forming focus for reflected light reflected from the mirror; a substrate part including two-photon photopolymerization resin on the sub strate for forming a micro/nano-structure; a stage for transporting the substrate to X, Y and Z axis; a CCD camera extracting fluorescence force created in the resin on the substrate; and a control computer automatically controlling the location of the stage for moving the substrate to a searched location by searching the focus location automatically using fluorescence force extracted by the CCD camera.
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