We propose a method capable of focusing a laser beam on a substrate automatically via fluorescence detection from the resin of a two-photon nanofabrication system. When two-photon absorption (TPA) occurs by focusing the laser beam in the resin, fluorescence is emitted from the focusing region in the visible range. The total pixel number above the threshold value of the fluorescence images obtained by a CCD camera is plotted on a graph in accordance with the focus position. By searching for the position when the total pixel number undergoes an abrupt change in the pre-TPA region, the correct configuration of the focused laser beam can be found. Through focusing tests conducted at four vertices of a 500 μm x 500 μm square placed arbitrarily inside SCR500 resin, the errors of the autofocusing method were found to range from -100 nm to + 200 nm. Moreover, this method does not leave any polymerized marks. To verify the usefulness of the autofocusing method, the fabrication of a pyramid structure consisting of 20 layers was attempted on a coverglass. It was completely fabricated without losing a layer.