Understanding the interactions between heterogeneous dissolved organic matter (DOM) and nonylphenols (NPs) is essential for predicting their behavior and fate in the environment. Herein, we firstly obtained different MW-fractionated humic acids (HAs) using the ultrafiltration method. Afterward, the molecular weight (MW)-dependent interactions of HAs with 4-nonylphenol (4-NP) were analysed by excitation emission matrix (EEM) fluorescence spectroscopy, fluorescence quenching, UV-vis spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and principal component analysis (PCA). EEM spectra indicated that the quenching mechanism was static. In the binding process, the higher MW fractions showed stronger interaction with 4-NP than the lower MW counterparts, exhibiting a clear MW-dependent interaction heterogeneity. The interaction constants for the 4-NP-HAs system were suppressed as the ionic strength decreased and pH increased, which was especially obvious in the binding of 4-NP to the lower MW-fractionated HAs. The FTIR spectra revealed that hydroxyl and aromatics were involved in the interaction process of HA fractions with 4-NP. It was also found from 1 H NMR that π-π interactions between aromatic rings of 4-NP and MW-fractionated HAs were responsible for the complexation. The correlation analysis and PCA results indicated that aromaticity and MW play important roles in the interaction process and confirmed an obvious interaction heterogeneity among MW-fractionated HAs samples. This work highlighted MW-dependent interaction heterogeneities of HA, which suggested that heterogeneity in MW distribution should be taken into consideration when exploring the fate and biogeochemistry cycling of 4-NP from contaminated environments.