Urban non-point source pollution contributes large amounts of dissolved organic matter (DOM) to aquatic ecosystems and has a significant impact on ecosystem health. Chromophoric dissolved organic matter (CDOM) is a group of macromolecular polymers with complex composition and structure, containing a variety of highly reactive chemical functional groups, which are widely distributed in natural waters. It has an important impact on aquatic ecosystem health, energy flow and biogeochemical cycling. Photochemical reactions and microbial metabolic processes are the main controlling factors on the transformation, degradation and cycling of CDOM in water bodies. However, understanding how urbanization source input impacts CDOM quantity and quality and how photochemical and microbial interact to affect CDOM dynamic are deficient. To assess the contribution of photochemical processes and microbial metabolism to the degradation and transformation of different sources of CDOM in urban waters, and to elucidate the mechanisms of photochemical/microbial degradation of different sources of CDOM in aquatic systems (non-urban lake with humic, mostly terrestrial CDOM; urban lake with autochthonous biological production CDOM; urban river with mixed humic and protein-like CDOM), this study compares and analyses the changes in the sources and composition of CDOM under the influence of photochemistry and microorganisms in a controlled 9-day laboratory incubation experiment in three water bodies with typical urban characteristics, selected in Birmingham, UK. Changes in CDOM concentration, optical properties and chlorophyll concentration were measured at the initial and end of incubations. The results show that: (1) due to the acceptance of upstream sewage discharge and shorter hydraulic retention times, the CDOM of urban rivers is dominated by aromatic substances, with the highest photochemical degradation rate of 16.60%; (2) the CDOM of urban lakes, which is affected by human activities, is dominated by autochthonous substances, and could be increased by 62.16% via microbial metabolism, while compared to urban lakes, non-urban lakes have a more pronounced effect of light on their degradation and transformation due to receiving a large input of humus-like CDOM from the surrounding landscape soils; (3) the photochemical process could degrade terrestrial humic-like substances into small molecular compounds with high biological activity, which could stimulate microbial metabolism to produce protein-like CDOM, however, photochemical degradation could inhibit microbial metabolism by transform the autochthonous protein-like CDOM into a refractory state. The study provides a novel perspective for the differences in the sources and activities of CDOM in different urban aquatic systems and the changes in CDOM characteristics during photochemical and microbial processes.