2D epitaxial nanostructures formed by functional molecules at interfaces/surfaces have attracted considerable research interest because of their promising applications in catalysis, photonics, electronic devices, and so on. In this study, we demonstrate for the first time that a monolayer of functional molecules may be used as a fluorescence indicator for heavy metal ions. Self-assembly of bisligand TPE-C4-L2 on highly oriented pyrolytic graphite was performed and the self-assembled monolayer (SAM) was used for trace amounts of Zn(II) sensing with an output signal of fluorescence. In contrast to the Zn(II)-induced fluorescence enhancement of the TPE-C4-L2 block molecule in solution, the self-assembled TPE-C4-L2 film showed quenched fluorescence after Zn(II) was introduced. Using a high-resolution scanning tunneling microscope, the SAM structures of TPE-C4-L2 and its zinc complex were determined. The correlation between their optical properties and structures at the molecular level was also revealed.