Steroid estrogens, such as 17β-estradiol (E2), in animal manure pose a potential threat to the aquatic environment. The transport and estrogenicity of estrogens influence the sorption of estrogens to dissolved organic matter (DOM) in animal manure, and composting treatment alters the structure and composition of the manure. The objectives of the present study were to identify the contribution of the molecular composition of DOM of composted manure to the sorption of E2 and then elucidate the dominant mechanisms involved in the interaction of E2 with manure-derived DOM. The excitation–emission matrix (EEM) spectra and atomic force microscopy (AFM) showed that composting significantly altered the chemical composition and structure of DOM. A decrease in the atomic ratios of oxygen (O)/carbon (C) occurred in conjunction with the formation of DOM aggregates in the composted manure, indicating that the hydrophilicity and polarity of the DOM decreased after composting. Composting increased the sorption coefficients (KDOC₋E₂) for E2 to DOM, and KDOC₋E₂ was positively correlated with the proportion of the fulvic acid (FA)-like fraction and molecular weight (MW) fractions of the DOM (range of 1.0 × 10³–7.0 × 10³ Da and 7.0 × 10³–1.4 × 10⁴ Da). Specifically, E2 showed a tendency for sorption to medium-sized FA-like molecules of DOM aggregates in composted manure. Hydrophobic forces and π-π binding appeared to be the main mechanisms underlying the aforementioned interaction.