Membrane fouling is still a significant drawback of membrane technology. Irreversible fouling can be alleviated only by chemical cleaning. Guidelines for determining the methods and efficiency of chemical cleaning, such as those for timing and chemical category, are still not available because of the lack of knowledge about foulants during membrane filtration. In this study, a new in situ observation method, i.e., three-dimensional solid-phase fluorescence excitation–emission matrix spectroscopy (SPF-EEM), was used to characterise the chemical cleaning efficiency of irreversible fouling. Caustic, acidic and hypochlorite chemical solutions were tested as cleaning agents used in different sequences. Progression trends of irreversible fouling were then characterised by in situ and online SPF-EEM. Chemical cleaning efficiency observed by SPF-EEM was consistent with that observed by conventional infrared spectroscopy analysis, which only can be conducted ex situ. By performing SPF-EEM analysis, the optimal chemical cleaning procedure, which exhibited a result consistent with that obtained for the recovery of pure water permeability, could be determined. Hence, SPF-EEM is effective for the in situ characterisation of irreversible fouling, and it is beneficial for the determination of the timing and chemical category for cleaning based on the SPF-EEM of foulants.