Arsenic (As) can form complexes with dissolved organic matter (DOM), which affects the fate of arsenic in waste sites and natural environments. It remains a challenge to analyze DOM-bound As, in particular by using a direct chromatographic separation method. Size exclusion chromatography (SEC) hyphenated with UV spectrophotometer and inductively coupled plasma mass spectrometry (ICP-MS) was developed to characterize the complexation of arsenite (As(III)) with DOM. This SEC-UV-ICP-MS method is able to differentiate As(III)-DOM complexes from free As species and has the advantage of direct determination of both free and DOM-bound As(III) through mild separation. The suitability of this method for studying As(III)-DOM complexation was demonstrated by its application, in combination with the Scatchard plot and nonlinear regression of ligand binding model, for characterizing As(III) complexation with humic acid (HA) in the absence or presence of natural sand. The results suggest that, consistent with polyelectrolytic nature of HA, the As(III)-HA complexation should be accounted for by multiple classes of binding sites. By loosely classifying the binding sites into strong (S1) and weak (S2) sites, the apparent stability constants (Ks) of the resulting As-DOM complexes were calculated as logK(s1) = 6.5-7.1 while logK(s2) = 4.7-5.0.