Zucchini yellow mosaic virus (ZYMV) is a significant pathogen causing yellow mosaic disease in Cucurbitaceae. It can spread rapidly from infected plants to healthy ones or through contaminated seed sources, leading to a substantial reduction in the yield and quality of pumpkins after harvest. Currently, there is no effective treatment to eliminate this virus, making seed screening prior to planting and the removal of symptomatic plants the most effective control methods. In this study, a 214 bp target gene of the ZYMV was amplified using specific primers, then cloned into the pJET1.2 vector and transformed into Escherichia coli JM109. A realtime RT-PCR procedure was developed to detect and quantify ZYMV utilizing a primer pair designed for a 164 bp product. The standard curve was established with the equation y = -3.417x + 49.605 and correlation coefficient R2 = 0.9969 for quantifying the ZYMV virus. The realtime RT-PCR was built with qualitative results corresponding to the PCR method. Additionally, the procedure quantified test samples with viral loads ranging from 7.1 x 106 to 8.5 x 109 copies/μL.