This study was aimed at comparing the Zn and Cu binding parameters with dissolved organic matter (DOM) in road runoff, wastewater treatment plant (WWTP) effluent and influent. Conditional stability constant (K´) and binding site concentration ([L]) are important to predict free or labile metal concentration and toxicity in the water environment. The values of K´ and [L] of three DOMs were determined by metal titration and Scatchard linearization. The Zn and Cu titration data for DOMs in WWTP effluent and influent fitted to a two-ligand model, while DOM in road runoff fitted to a single-ligand model. The order of the values of K´ was WWTP influent > WWTP effluent > road runoff both for Zn and Cu. Total ambient binding site concentration ([LT]) was lower in DOM of road runoff (1.4–1.5 mM) than those in DOM of WWTP effluent (1.6–2.3 mM) and influent (17–18 mM), suggesting lower metal complexation capacity in DOM of road runoff. DOM in WWTP influent was expected to bind to both Zn and Cu more strongly than that of the effluent DOM.