The pore size of an anodic aluminum oxide (AAO) chip, as well as uniform pore distribution, is one of the key parameters that should be adjusted, by choosing the appropriate etching conditions, in order to enhance the sensitivity of an interferometer. In this study, the pore size of AAO chips was optimized and characterized in order to lower the detection limit of prostate specific antigen (PSA) in an interferometric immunoassay system. After pore widening for 30-50 min, the AAO pore size was increased approximately 2-fold larger than that before pore widening. A large increase in effective optical density (ΔEOT) was obtained from the AAO chip fabricated by pore-widening technique, which thereby lowered the PSA detection limit. The present study results are not sufficiently validated to enable the immediate application to immunoassay for prostate cancer (PCa) screening, but they do demonstrate that controlling pore size can positively affect the sensitivity and lower the detection limit.