Fluorescence of molecules on metal substrates has attracted much attention because of its relevance to various optical devices, such as light-emitting diodes, sensors, and solar cells. However, the fluorescence measurement near the metal substrates is challenging due to the rapid quenching of the excited state of the molecules. In this study, fluorescence of tetraphenylporphyrin (TPP) on Au(111) substrates was successfully measured by fabricating a mixed self-assembled monolayer (SAM) of the tripodal molecules with and without TPP dye. The rigid tripodal molecules suppressed quenching, prevented structural changes, and reduced interactions between TPP moieties. These features made it possible to measure the fluorescence of TPP molecules isolated and dispersed on Au(111) in a uniform and well-defined structure. Through excitation spectra acquired by sweeping the excitation wavelength, information about TPP absorption was also obtained. In addition, these measurements were possible under potential control in an electrochemical environment due to the stability of the SAMs. Thus, we demonstrated that the system based on tripodal molecules can serve as a platform for measuring the fluorescence and absorption of molecules on metal substrates in the atmospheric and electrochemical environments in which various optical devices operate.