Coupling of anaerobic ammonium oxidation (anammox) and denitrifying anaerobic methane oxidation (DAMO) microorganisms in a hollow-fiber membrane biofilm reactor (HfMBR) is a potential strategy for simultaneous anaerobic removal of nitrogen and methane in wastewater streams. In these systems, effluents contain dissolved organic substances from anammox and DAMO microorganisms, but their characteristics and relationships have not been investigated. In the present study, excitation-emission matrix (EEM) fluorescence spectroscopy was used to characterize effluent dissolved organic matter (EfDOM) from an Anammox-DAMO HfMBR. Four component types (Component 1-4) were identified by parallel factor analysis (PARAFAC) of EEM data. Component 1 was produced when anammox and DAMO microorganisms simultaneously starved, whereas Component 4 was only generated through the starving period of DAMO microorganisms, and the longer the starving period, the higher the fluorescence intensity of the components. Components 2 and 3 were generated via active and starving periods of co-cultures. More efficient nitrogen removal was accompanied by a higher fluorescence intensity and microbial activity. Compared to measuring both influent and effluent nitrogen concentrations, monitoring EfDOM can obtain other information about the reactor, such as nitrogen removal activity of the reactor, status of the microbes and the duration of starving period the reactor suffered, which therefore offers a complementary but direct tool for assessing reactor performance in complex co-culture systems.