Chitosan-functionalized amino acids offer a sustainable approach to plant biostimulation. This study developed a novel and green method to synthesize mono- and dual-functionalized chitosan matrices incorporating L-Tryptophan, L-Valine, and l-Lysine—amino acids known for their biostimulant properties. The resulting compounds included Val-chitosan, Trp-chitosan, Val.Lys-chitosan, and Trp.Lys-chitosan. Structural characterization was performed using FT-IR and NMR spectroscopy. The FTIR spectra showed distinct absorption bands of interest, including the NH stretch (3300–3400 cm-1), CH stretch (2800–3000 cm⁻¹), and amide bonds around 1650 cm⁻¹. ¹H NMR analysis provided detailed structural groups such as tryptophan's aromatic protons, methylene protons of lysine side chains, and valine's isopropyl groups, indicating successful stepwise grafting. The biostimulant efficacy was evaluated through laboratory trials on barley plants (Hordeum vulgare L. cv. Olsok), comparing treated and untreated groups. The dual-functionalized Trp.Lys-chitosan significantly enhanced growth, with stem length increasing by 471 % after 15 days and 354 % after 30 days, and root length expanding by 300 %. Val.Lys-chitosan improved leaf diameter, internode length, moisture content, and boosted photosynthetic efficiency by 15.6 %.