Dissolved organic matter (DOM) plays a major role in ecological systems and influences the fate and transportation of many pollutants. Despite the significance of DOM, understanding of how environmental and anthropogenic factors influence its composition and characteristics is limited, especially in urban stormwater runoff. In this article, the chemical properties (pollutant loads, molecular weight, aromaticity, sources, and molecular composition) of DOM in stormwater extracted from three typical end-members (traffic, residential, and campus regions) were characterized by UV–visible (UV–vis) spectroscopy, excitation-emission matrix spectroscopy combined with parallel factor analysis (EEM-PARAFAC), and ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). There are three findings: (1) The basic properties of DOM in stormwater runoff varied obviously from three urban fields, and the effect of initial flush was also apparent. (2) The DOM in residential areas mainly came from autochthonous sources, while allochthonous sources primarily contributed to the DOM in traffic and campus areas. However, it was mainly composed of terrestrial humic-like components with CHO and CHON element composition and HULO and aliphatic formulas. (3) The parameters characterizing DOM were primarily related to terrestrial source and aromaticity, but their correlations varied. Through the combination of optical methods and UPLC-Q-TOF spectrometry, the optical and molecular characteristics of rainwater are effectively revealed, which may provide a solid foundation for the classification management of stormwater runoff in different urban regions.