In recent years, developments of drug delivery systems of antibiotics have been needed for improving antibacterial performance due to massive usage of antibiotics. With the global growing concept of being green, lignosulfonate (LS) has been deemed as an ecofriendly stabilizer and carrier that holds promise in preparing nanocomposites for antibacterial treatment. At the same time, LS and gentamicin sulfate (GS) have been proven to form a nanocomposite (LS-GS) with highly uniform size and roundness in solution. It also possesses a good loading capacity of GS. Density functional theory (DFT) verified that the interaction forces in LS-GS, a nanocomposite with selfassembly characteristics, were mainly combined via hydrogen bonds, van der Waals forces, and repulsion. The obtained LS-GS has the biocompatible, biodegradable, and low-cost merits of LS. It also exhibited excellent in vitro antibacterial activities against Escherichia coli and Staphylococcus aureus due to the antibacterial properties of GS. Moreover, an in-depth antibacterial study revealed the highly efficient antibacterial and woundhealing applications of LS-GS in vivo. Collectively, LS, a carrier material with low cost, which strikingly increases the antibacterial and wound-healing ability of GS in vitro and in vivo, will contribute to the production of large-scale antibacterial nanocomposites.