The basic-leucine zipper (bZIP) transcription factor (TF), which is abundant and highly conserved in eukaryotes, mainly participates in plant growth, development and adverse stress processes. However, the regulatory mechanisms of bZIP TFs under heat stress in Dimocarpus longan remain unclear. In this study, we reported a nuclear-localized bZIP TF, DlbZIP20, which was confirmed to be a positive regulator of thermotolerance in D. longan. It was identified as a strong interplay with hormone signalling pathways and oxidoreductase-related families. The transient overexpression DlbZIP20 could improve the thermotolerance of D. longan embryogenic callus (EC) and reduce the damage of cells under heat stress. Overexpression of DlbZIP20 in hairy roots significantly promoted roots elongation and photosystem II (PS II) responses in leaves, and significantly increased SOD and POD activities, promoted H