Abstract: To investigate the role of the plant sulfopeptin (PSK) small peptide in tobacco drought resistance, we utilized bioinformatic methods to identify 9 NtPSK genes within the tobacco genome and analyze their corresponding protein structures. Subsequently, PSK peptide was administered to tobacco plants under drought conditions, to evaluate its effects on the development of tobacco seedlings and plants. Our findings revealed a highly conserved YIYTQ motif in the C-terminal region of the 9 prePSK proteins. Notably, treatment of tobacco seedlings with 1 μmol/L PSK peptide during drought stress promoted taproot elongation, substantially enhanced leaf SOD activity, and reduced reactive oxygen species levels. Similarly, application of 1 μmol/L PSK peptide to tobacco plants yielded significant improvements in drought resistance, evidenced by increased relative water content and proline levels, alongside notable reductions in malondialdehyde and hydrogen peroxide content. These findings collectively suggest that PSK peptide holds promise in alleviating the deleterious effects of drought on tobacco growth and development, thereby enhancing its resilience to drought stress.