Dissolved organic matter (DOM) will be increasingly monitored by means of in situ fluorescence spectroscopy devices in order to supervise wastewater treatment plant efficiency, due to their ease of implementation and high-frequency measurement capacity. However, fluorescence spectroscopy measurements are reported to be sensitive to the sample matrix effects of temperature, the inner filter effect (IFE), and turbidity. Matrix effect estimation tests and signal correction have been developed for DOM (tyrosine-like, tryptophan-like, and humic substances-like fluorescent compounds) fluorescence measurements in unfiltered urban sewage samples. All such tests are conducted in temperature, absorbance, and turbidity ranges representative of urban sewage. For all fluorophores studied, an average of 1% fluorescence intensity decrease per degree (degrees C) of temperature increase could be observed. Protein-like fluorescent compound signals were found to be significantly affected by turbidity (0 to 210 NTU) and IFE (absorbance 254 nm > 0.200). Only temperature needs to be corrected for humic substances-like fluorescent compounds since other effects were not observed over the studied ranges of absorbance and turbidity. The fluorescence intensity correction method was applied first to each matrix effect separately and then combined by using a sequential mathematical correction methodology. An efficient methodology for determining the matrix effect correction equations for DOM fluorescence analysis into unfiltered urban sewage samples has been highlighted and could be used for in situ fluorescence measurement devices.