In the past decade, the exploration of genetic resources in rice has significantly enhanced the efficacy of rice breeding. However, the exploration of genetic resources is hindered by the identification of candidate genes. To expedite the identification of candidate genes, this study examined tapetum programmed cell death-related genes OsiWAK1, OsPDT1, EAT1, TDR, and TIP2 to assess the efficacy of the Dual-Luciferase (Dual-LUC) assay in rapidly determining gene relationships. The study found that, in the Dual-LUC assay, OsiWAK1 and its various recombinant proteins exhibit comparable activation abilities on the EAT1 promoter, potentially indicating a false positive. However, the Dual-LUC assay can reveal that OsiWAK1 impacts both the function of its upstream regulatory factor OsPDT1 and the TDR/TIP2 transcription complex. By rapidly studying the relationship between diverse candidate genes and regulatory genes in a well-known trait via the Dual-LUC assay, this study provides a novel approach to expedite the determination of candidate genes such as genome-wide association study.