The spatial and temporal dynamics of chromophoric dissolved organic matter (CDOM) were studied using excitation emission matrix fluorescence spectroscopy (EEMs) and parallel factor analysis (PARAFAC) during five cruises in the subtropical Jiulong Estuary from August 2008 to June 2009. Two humic-like (C1 and C3), one tryptophan-like (C4) and one possible protein-like (C2) component was identified by PARAFAC and their behavior in the river–estuary–coastal interface was evaluated. The spatial distributions of the maximum fluorescence (Fmax) for the fluorescent components showed a remarkable loss in the upper estuary. The following significant addition of all components in the low salinity turbidity maximum zone suggested the inputs from riverine source, sediment resuspension and the surrounding mangrove ecosystem. C1, C2 and C3 showed conservative behavior in the middle and lower estuary indicated by the linear relationship between their fluorescence intensities and salinity in the five cruises. However, the tryptophan-like C4 received widespread additions (likely from autochthonous production) in the estuary. Although the humic-like C1 and C3 showed no significant variation in the estuary–coastal interface, C2 and C4 decreased more rapidly beyond this interface, indicating the significant influence of coastal current to this estuarine environment. The seasonal variation of tryptophan-like C4 was characterized by higher Fmax values in the upper to middle estuary in the dry season, which is in contrast to that of the humic-like C3. Correlation analysis with DOC showed that the fluorescence intensity of C1 (or C2 and C3) was suitable for tracing DOC dynamics in this estuary. These results indicated different mixing behavior and temporal variability for different fluorescent components in this dynamic estuarine environment.