Decaying algal blooms in eutrophic lakes can introduce organic matter into the water and change nutrient concentrations in the water column. The spatial distribution and composition characteristics, sources, and contribution to eutrophication of dissolved organic matter (DOM) in the overlying water of Lake Taihu, a typical eutrophic lake in China, were analyzed by ultraviolet–visible spectra and three-dimensional fluorescence excitation–emission matrix spectra combined with the statistical decomposition technique, parallel factor analysis. The concentration of DOM was represented by dissolved organic carbon (DOC), and DOC in overlying water of Lake Taihu was 2.86–11.83 mg/L. The colored DOM (CDOM) was characterized by an absorption coefficient at 280 nm (a280) and 350 nm (a350), which were 6.63–29.87 and 1.84–10.41 m−1, respectively. These values showed an increasing trend from southeast to northwest, and the high values were concentrated in the northwest and northern lake areas. The parallel factor analysis (PARAFAC) identified two protein-like (C1: tyrosine-like and C2: tryptophan-like) and one humic-like (C3: humic acid and fulvic acid) fluorescence components for fluorescent DOM (FDOM). The most dominant components were protein-like components (C1 + C2), whose fluorescence intensity contributed 87.55% ± 3.39% to the total fluorescence intensity (Ft) of FDOM (3.38 R.U.). The mean value of the fluorescence index (FI) and index of recent autochthonous contribution (BIX) of DOM was 1.77 and 0.92, and DOC, a280 and fluorescence intensities of FDOM components were all significantly and positively correlated with chl. a, indicating that DOM, CDOM, and FDOM were all mainly derived from algal activities and metabolites. The average humification index of the DOM was 0.66, which indicated a low humification degree. The protein-like DOM was correlated with DON and DOP, and might make great contributions to the continuous occurrence of algal blooms.