Background <p>The DNA-binding with one finger (<i>Dof</i>) gene is a classical plant transcription factor with specific conserved structural domains, which plays an important role in the regulation of plant metabolism, root development, and stress defense mechanisms. In recent years, drought has severely jeopardized the growth and development of crops and is one of the main environmental factors limiting plant growth and reducing crop yields. Whether <i>Dof</i> family genes play a role in mulberry drought resistance is largely unknown.</p> Results <p>To investigate the characteristics of the <i>Dof</i> gene family members in mulberry trees, 24 members of the mulberry <i>Dof</i> gene family were identified in the whole mulberry genome, and bioinformatics tools were employed to analyze the protein physicochemical properties, gene structure, phylogenetic evolution, conserved sequences, interaction networks, promoter cis-acting elements, and expression levels of <i>Dof</i> genes in mulberry.</p> Conclusions <p>Most of the mulberry Dof proteins were unstable, all were hydrophilic. Subcellular localization prediction showed that all proteins were localized in the nucleus. The prediction of promoter cis-elements showed that the <i>MnDof</i> (<i>Morus notabilis</i> L. <i>Dof</i>) gene family contained 75 promoter cis-elements for growth and development, hormone, light, and stress responses. Amino acid sequence alignment and phylogenetic tree analysis showed that all 24 <i>Dof</i> transcription factors contained zinc finger structural proteins. <i>MnDof</i> genes were distributed on all chromosomes except chromosomes 1, 2, and 7. A portion of <i>MnDof</i> genes responded to drought stress. These results provide information on the <i>Dof</i> gene family in mulberry and provide new insights into the function of <i>MnDof</i> genes in perennial plants, laying the foundation for further analysis of the function of these genes.</p>