Understanding the interaction between biocides and heavy metals with dissolved organic matter (DOM) is the basis of building runoff treatment facility development. In the present study, we aimed to quantitatively determine the binding of biocides/heavy metals with DOM and evaluate the effect of Cu2 + on biocides-DOM interaction. We hypothesize that biocides prefer to bind with DOM having a larger molecular weight (MW), and this binding can be inhibited by Cu2+. In experiments, DOM was separated into different fractions according to their MW difference, then we investigated the binding of Cu2+, Zn2+, biocides, and their transformation products (TPs) to different DOM fractions by dialysis equilibrium. According to the results, Cu2+ was found having the highest affinity for DOM fraction > 100 kDa with the mean binding value of 2.65 μmol/mgC, which reduced with decreasing DOM MW. Zn2+ binding to DOM was much weaker than Cu2+ with a mean value below 0.296 μmol/mgC and Zn2+ did not show a clear preference for DOM MW. Parent biocides showed higher DOM affinity than their TPs. The binding affinity of biocides to DOM followed the order mecoprop-p > terbutryn > diuron. Hydrogen bonding was found to play an important role in biocides-DOM binding. Moreover, the opposite effect of Cu2+ on biocides-DOM binding was observed, with inhibition for DOM fractions> 100 kDa and enhancement for DOM fractions < 100 kDa. Our findings suggest that source control of biocides/TPs pollution is important because a relatively high mobility of biocides/TPs in the environment is expected due to their limited interaction with DOM.