Pathogenesis-related protein 1 (PR-1) genes are involved in plant defense responses and abiotic stress responses. In this study, a total of 14 PfPR-1 family members were identified from the genome of Paulownia fortunei, and their physicochemical properties, phylogenetic relationships, conserved motifs, promoter cis-acting elements, chromosomal localization, and syntenic relationships were systematically analyzed. The results showed that PfPR-1 family members exhibited considerable variation in protein physicochemical characteristics and structural composition, suggesting evolutionary and functional divergence among different members. Promoter analysis revealed the presence of numerous hormone-responsive, stress-responsive, and light-responsive cis-acting elements, indicating that PfPR-1 genes may be involved in multiple biological processes and signaling pathways. Transcriptome sequencing analysis demonstrated that most PfPR-1 genes significantly responded to Paulownia witches’ broom (PaWB) disease, salt stress, and drought stress. Under phytoplasma infection, five PfPR-1 genes were markedly upregulated (PR-1-1, PR-1-3, PR-1-4, PR-1-8, and PR-1-14), whereas four PfPR-1 genes (PfPR-1-6, PfPR-1-7, PfPR-1-12, and PfPR-1-13) were downregulated, suggesting their differential responses to phytoplasma infection. Under salt- and drought-stress conditions, different PfPR-1 genes exhibited diverse expression patterns, indicating their potential functional specialization in stress-responsive regulation. Subsequent qRT-PCR analysis further confirmed the reliability of the transcriptome data. Notably, the expression of PR-1-3 was significantly upregulated under both biotic and abiotic stresses. Yeast one-hybrid and dual-luciferase reporter assays demonstrated that an ERF transcription factor (TF), PfERF1, could significantly activate the transcription of PfPR-1-3, suggesting that PfERF1 may participate in stress-responsive regulation in Paulownia fortunei through modulating PfPR-1 expression. Collectively, these findings provide a theoretical basis for further investigation of the biological functions and molecular regulatory mechanisms of the PfPR-1 gene family during stress responses in Paulownia fortunei.