Effluent organic matter (EfOM) is one of the major foulants responsible for the occurrence of membrane fouling during advanced wastewater treatment using nanofiltration (NF) technology. In this present study, we have reported a simple fouling indicator based on the properties of fractional fluorescence and molecular weight, termed as fluorescence-size-index (FSI), to predict the fouling propensity of NF when filtrating EfOM. Specifically, EfOM collected from twenty-one real sewage samples were first analyzed to quantify their fluorescent compositions and concentrations. The results showed that the EfOM consisted mainly of humic-like substances, soluble organism metabolites and fulvic-like substances, characterized by small-molecule organic matters (< 5 kDa) and hydrophobic fractions. Second, the major NF fouling fractions of EfOM were determined based on their fluorescent properties. It was observed that small-molecule hydrophobic components with humic-like fluorescence properties continuously influenced the flux decline rate throughout the whole operation, while macromolecular hydrophilic components with fluorescent properties of apparent aromatic hydrocarbon proteins were primarily responsible for the initial, rapid flux decline. Furthermore, the constructed FSI has proven to be useful in guiding the selection of pretreatment methods for preventing NF fouling.