The fluorescence spectroscopy is an analytical tool used to test water from different origins because it is sensitive, selective and can give a broad spectrum of information on the composition, characteristics, origin and distribution of dissolved organic matter (DOM). The objective of this research was to characterize the effluent DOM of an aerobic autotrophic nitrifying reactor with fluorescence spectrometry of the excitation-emission matrix in 3D (EEM-3D) and determine if ·EEM-3D could identify soluble microbial products from nitrifying bacteria. The nitrifying reactor was operated at a sludge retention time of 36 days, a hydraulic retention time of 40 hours of hydraulic retention time and fed with an excess of ammonium. Only two fluorescence peaks were identified in the reactor effluent: The peaks were located at similar emission wavelength (416.9 ± 10.3 and 415.5 ± 2.2 nm, respectively), but different excitation wavelength (245.2 ± 1.0 and 330.7 ± 1.7 nm, respectively). The EEM-3D were significantly different than those found in the effluent of aerobic and anaerobic reactors.