The characteristics of fluorescent components of dissolved organic matter (DOM) were examined using excitation emission matrix (EEM) fluorescence spectroscopy combined with parallel-factor analysis (PARAFAC) for seawater samples obtained from the northern Gulf of Mexico (NGoM) before, during, and after the 2010 Deepwater Horizon (DwH) oil spill. An EEMs PARAFAC modeling of samples collected within 16 km of the wellhead during the oil spill in May 2010, which included one typical subsurface sample with a PAH concentration of 1.09 μg/L, identified two humic-like and two previously reported oil-like components. Compared to prespill levels, however, there were order-of-magnitude higher fluorescence intensities associated with these components that are consistent with an oil-spill source. The spectral decomposition of the EEMs data using individual and combined data sets from coastal and offshore waters impacted by the DwH spill further revealed the changing nature of fluorescent DOM composition. Although the PAHs concentrations were at prespill conditions after the spill in 2012 and 2013 near the DwH site, the variable and anomalous levels of fluorescence intensities and DOC concentrations three years after the spill suggest the potential long-term persistence of the oil in the DOC pool in the NGoM.