BACKGROUND: SWEET (Sugar Will Eventually be Exported Transporter) proteins play vital roles in the transport of sugars, contributing to the regulation of plant development, hormone signaling, and responses to abiotic stress. RESULTS: In this study, we identified 57 NtSWEETs in tobacco (Nicotiana tabacum L.), and then their physicochemical properties, chromosomal localization, synteny, phylogenetic relationships, genomic structure, promoter cis-elements, protein interaction network, tissue specificity, and expression pattern were systematically analyzed. In addition, NtSWEET12i improves drought and saline-alkali tolerance in tobacco by enhancing soluble sugars transport, ABA signaling, proline biosynthesis, and ROS scavenging. These findings illuminate the role of NtSWEETs, particularly NtSWEET12i, in regulating plant tolerance to drought and saline-alkali stresses. CONCLUSIONS: This study offers new insights to enhance our understanding of the roles of NtSWEETs and identify potential genes involved in drought and saline-alkali tolerance of plants. CLINICAL TRIAL NUMBER: Not applicable.