Fusarium napiforme is a phytopathogenic fungus that causes substantial economic losses in various crops worldwide. Accurate and timely identification of this pathogen is crucial for effective disease management strategies. The present study performs a molecular identification method using polymerase chain reaction (PCR) to detect and identify Fusarium napiforme. In this study, specific primers targeting the internal transcribed spacer (ITS) region of the fungal rRNA gene were designed and used in PCR amplification. Fusarium napiforme was isolated from ridge gourd (Luffa acutangula) seeds by agar plate method. DNA was extracted from the isolates and subjected to PCR using the designed primers. The resulting PCR products were then analyzed by gel electrophoresis to determine the presence or absence of Fusarium napiforme. The PCR assay demonstrated high specificity and sensitivity in detecting Fusarium napiforme. The designed primers exclusively amplified the target DNA fragment from Fusarium napiforme isolates. The PCR method provides a rapid and reliable tool for the accurate identification of Fusarium napiforme. This technique can significantly aid in early disease diagnosis and facilitate prompt implementation of appropriate management strategies to control Fusarium napiforme infections. Additionally, it can assist in quarantine measures to prevent the spread of the pathogen to unaffected areas.