In this study, the wildtype Panax notoginseng was used as the experimental material. The transcription factor gene PnWRKY1 was isolated from the P. notoginseng by homologous cloning, and the overexpression vector pCAMBIA2300sPnWRKY1 was transferred into P. notoginseng cells by Agrobacterium tumefaciensmediated transformation system. The contents of total saponins and four important monomer saponins were detected, and the expression levels of enzyme genes in the biosynthetic pathway were analyzed by qRTPCR. It provides a theoretical basis for the establishment of a highly efficient regulation strategy for the biosynthesis of P. notoginseng saponins. The results showed that: (1) the transcription factor PnWRKY1 is 810 bp in length encoding 269 amino acids. (2) PnWRKY1 overexpression vector pCAMBIA2300sPnWRKY1 was successfully constructed. Six transgenic cell lines (T₁-T₆) with resistance to kanamycin were obtained by Agrobacterium transformation. PCR analysis of the kanamycin gene nptⅡ showed that all cell lines had a 450 bp specific band consistent with the expected length, indicating that six PnWRKY1 transgenic cell lines were obtained, which overexpressed PnWRKY1 successfully. (3) The expression levels of PnWRKY1 in six transgenic cell lines were very significantly higher than that in wildtype cell lines, and the T₃ cell line with the highest expression was 5.36 times higher than the wildtype cell line. (4) The total saponins biosynthesis in PnWRKY1overexpressed cell lines was significantly promoted, the contents of total saponins in T₁-T₆ were 2.46, 1.98, 2.67, 1.74, 2.54 and 1.98 times of the wildtype cell line, respectively. In six PnWRKY1overexpressed cell lines, the content of four monomer saponins (R1, Rg1, Re and Rb1) were increased to some content compared with wildtype cell line, especially the T₃ cell line, the content of monomer saponin Re was the highest (37.81 mg/g). (5) Compared with the wildtype cell line, the highest expression levels of key enzyme genes PnDS, PnSS and PnSE in the synthesis pathway of P. notoginseng saponins in the overexpressed PnWRKY1 cell line were increased by 3.1, 4.0 and 4.5 times, respectively. This study showed that the transcription factor PnWRKY1 may affect the synthesis of P. notoginseng saponins indirectly by regulating the expression of key enzyme genes in the biosynthetic pathway of P. notoginseng saponins. The results provide references for the establishment of a highly efficient regulation strategy about the biosynthesis of P. notoginseng saponins.