Nuclear factor Y (NF-Y) transcription factors play an essential role in regulating plant growth, development, and stress responses. Despite extensive research on the NF-Y gene family across various species, the knowledge regarding the NF-Y family in Ginkgo biloba remains unknown. In this study, we identified a total of 25 NF-Y genes (seven GbNF-YAs, 12 GbNF-YBs, and six GbNF-YCs) in the G. biloba genome. We characterized the gene structure, conserved motifs, multiple sequence alignments, and phylogenetic relationships with other species (Populus and Arabidopsis). Additionally, we conducted a synteny analysis, which revealed the occurrence of segment duplicated NF-YAs and NF-YBs. The promoters of GbNF-Y genes contained cis-acting elements related to stress response, and miRNA–mRNA analysis showed that some GbNF-YAs with stress-related cis-elements could be targeted by the conserved miRNA169. The expression of GbNF-YA genes responded to drought, salt, and heat treatments, with GbNF-YA6 showing significant upregulation under heat and drought stress. Subcellular localization indicated that GbNF-YA6 was located in both the nucleus and the membrane. Overexpressing GbNF-YA6 in ginkgo callus significantly induced the expression of heat-shock factors (GbHSFs), and overexpressing GbNF-YA6 in transgenic Arabidopsis enhanced its heat tolerance. Additionally, Y2H assays demonstrated that GbNF-YA6 could interact with GbHSP at the protein level. Overall, our findings offer novel insights into the role of GbNF-YA in enhancing abiotic stress tolerance and warrant further functional research of GbNF-Y genes.