While the filtering and accumulation effects of the extracellular matrix (ECM) on nanoparticles (NPs) have been experimentally observed, the detailed interactions between NPs and specific biomolecules within the ECM remain poorly understood and pose challenges for molecular-level investigations. Herein, we adopt molecular dynamics simulations to elucidate the impacts of methyl-, hydroxy-, amine-, and carboxyl-modified gold NPs on the cell-binding domains of fibronectin (Fn), an indispensable component of the ECM for cell attachment and signaling. Simulation results show that NPs can specifically bind to distinct Fn domains, and the strength of these interactions depends on the physicochemical properties of NPs. NP-NH exhibits the highest affinity to domains rich in acidic residues, leading to strong electrostatic interactions that induce severe deformation, potentially disrupting the normal functioning of Fn. NP-CH and NP-COO selectively occupy the RGD/PHSRN motifs, which may hinder their recognition by integrins on the cell surface. Additionally, NPs can disrupt the dimerization of Fn through competing for residues at the dimer interface or by diminishing the shape complementarity between dimerized proteins. The mechanical stretching of Fn, crucial for ECM fibrillogenesis, is suppressed by NPs due to their local rigidifying effect. These results provide valuable molecular-level insights into the impacts of various NPs on the ECM, holding significant implications for advancing nanomedicine and nanosafety evaluation.