Landscape urbanization broadly alter watersheds ecosystems, yet the impact of nonpoint source urban inputs on dissolved organic matter (DOM) amount, composition and source is poorly understood. To systematically examine how DOM optical index and composition varied with urbanization, a unique long term observation dataset (4 years) of fluorescence excitation emission matrices (EEMs) was collected from two types of waters: urban waters and non-urban waters. Two humic-like DOM fluorescent components (C1 and C2) and one protein-like component (C3) were identified by parallel factor analysis (PARAFAC), and the results indicated that urbanization had an important influence on DOM concentration and composition, with urban waters having a high degree of DOM variation due to different land use surrounding each body of water. Urban waters presented higher DOM content, CDOM absorption and DOM fluorescence intensity (FI), a greater proportion of protein-like (26%>21.3%), and less proportion of humic-like (51.9%<57.6%) than non-urban waters, were dominated by allochthonous inputs. Moreover, the long-term observation of the urbanized DOM's dynamics was conducted on monthly, seasonal and yearly timescales. The results reflected the response of DOM to regional climate. Higher DOM amount and FI appeared in the summer due to autochthonous production comes from algae growth and allochthonous input comes from rainfall. It also revealed that continuous increase in impervious artificial surfaces caused by urban expansion, contributed to the increase in DOM quantity and drove DOM composition to be more protein-like. Consequently, these findings filled the knowledge gap of the mechanism of land-water interaction on DOM properties in freshwater ecosystems.