After HSP70 binds to the J domain of the substrate and co-chaperone protein, ATP is hydrolyzed to ADP, and the nucleotide exchange factors (NEFs) promote the release of ADP. Under physiological conditions, the nucleotide exchange step is the rate-limiting step, which is accelerated by NEFs. In this study, the promoter of nucleotide exchange factor ZjFes1 was cloned, and its expression in tissues and under heat stress was studied to understand the regulatory mechanism of ZjFes1 and provide the molecular basis to study heat tolerance mechanism of seagrass. It was found that the promoter has common cis-acting elements in promoter and enhancer regions CAAT-box, as well as light response elements AE-box, Box 4 and TCCC-motif, a cis-acting regulatory element essential for the anaerobic induction of ARE, hormone response elements CGTCA-motif and TGACG-motif (MeJA response element), GARE-motif (gibberellin response element), TGA-element (auxin response element), a cis-acting regulatory element related to meristem expression CAT-box, and a cis-acting element involved in defense and stress responsiveness of TC-rich repeats. Two-week-old seedlings exhibited weak GUS activities in their cotyledons. In addition, the AtFes1A promoter was constitutively active in the anthers. After exposure to 38 °C for 2 h, the root tips of two-week-old seedlings were stained a strong blue. Heat-inducible activities of GUS were also observed in the cotyledons, roots, leaves, anthers, sepals and siliques.