In this study, we fabricated a light addressable potentiometric sensor (LAPS)-type penicillin sensor using the self-assembled monolayer (SAM) method and investigated its response characteristics. In addition, the application of the fabricated LAPS sensor to detect concentration distribution and speed of diffusion was also evaluated. A penicillinsensing membrane was made using the SAM method, and immobilization was confirmed by atomic force microscophy (AFM). The enzyme response characteristics of the fabricated sensor were 60 mV/decade within the penicillin concentration range from 0.1 mM to 10 mM. The characteristics of the 2-D image resulting from the enzyme reaction between penicillin and penicillinase were also investigated. Image detection used a line scanning sampling method and required approximately 10 min for 128 × 128 pixels over a 1 × 1 cm sensing area.