Fluorescence excitation-emission matrix (EEM) spectrophotometry was applied to 10 sample sites in six rivers in northeastern England, some of which were adversely impacted by sewage treatment works (STW) discharges, with the aim to investigate whether STW discharge has a significantly distinct fluorescence signature. Upstream, downstream, and STW discharge samples for two STWs demonstrated that treated sewage has a distinct fluorescence EEM, with high tryptophan and fulvic-like fluorescence intensities that are of approximately equal ratio. This signature could be seen in downstream samples. When all 10 sample locations were compared, two trend lines were apparent where STW impacted rivers plotted separately from the other sample locations. Fluorescence EEM signatures were compared to absorption at 254 nm and demonstrated to provide a better fingerprint of sewage-impacted water. It is suggested that fluorescence EEM spectrophotometry can provide a useful tool for the analysis of grab samples taken for both routine and investigative monitoring and has the potential for on-line monitoring of STW impacts on river systems.