In order to understand Si behavior and biodisponibility in soils and plants, we evaluated the use of PDMPO (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole) that was supposed to be a Si-specific fluorescence marker and to have a pH-dependent fluorescence. We studied the interactions between PDMPO and water-dissolved Si, Al and natural organic matter (humic acids, HA). Six systems with different HA, Si and Al concentrations were studied by fluorescence spectroscopy at pH 4, 7 and 9. The Al–PDMPO complex was characterized by infrared spectroscopy and the particle size distribution in solution was characterized by nano tracking analysis. We found that when usual pH buffers are not present, the PDMPO fluorescence was not pH dependent and was not Si-specific. In the PDMPO–Si–HA system, the PDMPO fluorescence signals were greatly enhanced, suggesting the formation of highly fluorescent ternary HA–PDMPO–Si groups. When Al was added to the system, the fluorescence was strongly quenched, suggesting the formation of low-fluorescence quaternary HA–PDMPO–Si–Al groups. The PDMPO fluorescence is therefore greatly sensitive to complexable metals and to natural organic matter and is therefore difficult to be applied for the quantification of Si or pH in a complex medium.