Two series of lignin (LN)-based adsorbents, namely, cross-linked lignin (LNEs) with different crosslinking densities and carboxymethyl cross-linked lignin (LNECs) with various degrees of carboxymethyl substitution, were prepared to remove ofloxacin (OFL), a popular fluoroquinolone (FQ) antibiotic, from water. LNEs and LNECs exhibited satisfactory performance in OFL adsorption. Both of them had high adsorption capacity (the maximum contribution of 0.828 mmol/g), good anti-interference to some inorganic salts, and efficient regeneration and reuse performance. The crosslinking density and degree of carboxymethyl substitution strongly affected the content and distribution of oxygen-containing groups in these LN-based adsorbents, which played important roles in OFL adsorption. The pH dependencies of the adsorption performance of LNEs and LNECs indicated the involvement of multiple adsorption mechanisms, including hydrogen bond, electrostatic attraction, π-π electron–donor–acceptor interactions, and negative charge-assisted hydrogen bond. Different mechanisms were dominant under various pH levels, in a near neutral pH, the synergistic effect of electrostatic attraction and π-π interaction allows LINEs and LINECs to reach maximum adsorption capacity. Five FQs with similar structures and their two sub structural analogs were compared in terms of adsorption behavior and electrostatic potential by density functional theory using quantum chemical calculation. FQs with secondary amino groups and low π electron cloud density readily bound to LN-based adsorbents. Hence, LNEs and LNECs were efficient and environment-friendly adsorbents.