The effluents from the anaerobic methane reactor were characterized by the EEM fluorescence spectra. The EEM fluorescence spectra and relative concentration of the main components were acquired by PARAFAC models. Results show that the main components of the effluents from the uninhibited reactor were protein, coenzyme NADH and F420. When the methane-producing reaction was inhibited, the production rate of methane decreased rapidly. Correspondingly, the fluorescence of F420 disappeared and the main components of the reactor were protein and NADH. The fluorescence intensity scores of protein from the diluted sample were remarkably correlated to the protein determination by chemical analysis and the correlation efficient was 0.93. The fluorescence intensity scores of F420 were correlated to the production rate of methane, which implicated that coenzyme F420 could be used to reflect the activity of methane production in a certain bioreactor. This work provides a new method to monitor the operation status of anaerobic methane-producing reactor.