Water chemistry and concentration of dissolved humic acid (DHA) have been reported affecting the binding properties of DHA with hydrophobic organic contaminants (HOCs). However, exact relationship between DHA conformation and its binding properties remains unclear. In this study, the effect of DHA concentration on its binding capacity (KDOC) with pyrene was investigated under different pHs and in the presence of Al3+ using dialysis bags. In addition, we studied the conformation of DHA under different conditions via atomic force microscopy (AFM) imaging, dynamic light scattering, and zeta-potential measurements. Increased KDOC was observed with decreasing pH or in the presence of Al3+. According to AFM imaging, aggregation was observed at pH 4, while individual molecules were observed at pH 11 because of the electrostatic repulsion between the charged molecules. In the presence of Al3+, significant aggregation was observed because of the charge screen effect. Therefore, inter-molecular interaction was facilitated at low pH or with the presence of Al3+, and a more organized molecular aggregate (such as micelle-like structure) could form, thus, enhancing KDOC. A significant decrease of KDOC with increasing DHA concentration was observed for all conditions. As DHA concentration increased, DHA molecular aggregation was promoted in a way reducing the number of sorption sites. Our results indicate that inter-molecular interaction enhances the sorption at lower DHA concentration, while reduces the number of the sorption sites as concentration increases.