Abstract: The aim of this study was to investigate the regulatory role of zebrafish (Danio rerio) notch1a and notch1b genes on hsp70 by using a dual luciferase reporter gene assay. The CDS sequences of zebrafish notch1a and notch1b genes were obtained through NCBI database search, and their intracellular domains (Notch1a/Notch1b intracellular domain, N1aICD/N1bICD) were cloned. Eukaryotic expression vectors, pCMV-N1aICD and pCMV-N1bICD, were constructed. The expressions of N1aICD and N1bICD were detected in human embryonic kidney cells (HEK293T) through Western Blot and subcellular localization. The zebrafish hsp70 promoter sequence was analyzed, cloned, and incorporated into the pGL3-hsp70-pro reporter gene, which was constructed and assayed for dual luciferase activity in HEK293T. Overexpression of notch1a and notch1b genes in HEK293T cells was performed, and changes in pGL3-hsp70-pro activity were detected using a dual-luciferase reporter gene assay. The results showed that the successful construction of eukaryotic expression vectors pCMV-N1aICD and pCMV-N1bICD. Western Blot analysis confirmed the normal expression of pCMV-N1aICD and pCMV-N1bICD, while the subcellular localization assay showed the normal expression in the nucleus of HEK293T cells. The dual-luciferase reporter gene showed that the pGL3-hsp70-pro reporter gene was active in HEK293T cells, exhibiting activity 3.7 times higher than that of the empty plasmid. Additionally, the pCMV-N1aICD and pCMV-N1bICD eukaryotic expression vectors significantly enhanced the activity of pGL3-hsp70-pro in HEK293T cells, showing increases of 4.9-fold and 5.1-fold compared to the control, respectively. The results indicated that zebrafish notch1a and notch1b genes play a substantial role in enhancing the expression of hsp70 gene. This provides experimental materials and a theoretical foundation for further investigation into the immune mechanism of Notch molecular in defense against infection and apoptosis through Hsp70.