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Abstract Despite the advancements in analytical techniques, there are still great challenges and difficulties in accurately and effectively quantifying and characterizing dissolved organic carbon (DOC) in environmental samples. The objectives of this review paper are (a) to understand the roles and variability of DOC along the water continuum; (b) to identify the constraints, inconsistences, limitations, and artifacts in DOC characterization; and (c) to provide recommendations and remarks to improve the analytical accuracy. For the first objective, we summarize the four ecological and engineering roles of DOC along the water continuum from source water to municipal utility, including nutrients and energy sources, controlling the fates of micropollutants, buffering capacity, and treatability and precursors of disinfection byproducts. We also discuss three major challenges in DOC analysis, including spatial and temporal variations, degradability and stability, and unknown structures and formulas. For the second objective, we review the procedures and steps in DOC analysis, including sampling in diverse environmental matrices, isolation of DOC fraction, storage and preservation techniques, and analyses on bulk chemical characteristics. We list and discuss the available options and evaluate the advantages and disadvantages of each choice. Last, we provide recommendations and remarks for each stage: sampling, isolation, storage, and analysis.