Localized surface plasmon resonance (LSPR) is a promising method for detecting antigen-antibody binding in label-free biosensors. In this study, the fabrication of a LSPR substrate with a gold nanodot array through the lift-off process of an alumina mask is reported. The substrate showed an extinction peak in its extinction spectrum, and the peak position was dependent on the height of the gold nanodot array, and the change of extinction peak with the height could be predicted by the numerical simulation. In addition, the peak position was observed to be red-shifted with the increasing RIU value of the medium surrounding the gold nanodot array. In particular, the peak position in the 10 nm thick gold nanodot array was approximately 710 nm in air, and the sensitivity, defined as the ratio of the shift of peak position to the RIU of the medium, was 323.6 nm/RIU. The fabrication procedure could be applied to fabricate the LSPR substrates with a large area.