Background: Banana Fusarium wilt inflicts enormous economic losses on global banana production; however, effective control strategies remain elusive. We previously demonstrated that overexpression of plantain (Musa paradisiaca) ICE1 (MpICE1) significantly enhances banana resistance to Fusarium wilt. This study aimed to screen and identify key resistance genes associated with banana Fusarium wilt resistance and further explore their molecular functions and regulatory mechanisms, so as to provide a solid theoretical basis for the genetic breeding and disease resistance improvement of banana. Methods: Based on our previous transcriptomic dataset and the current metabolomic analysis, we identified a key gene encoding 12-oxo-phytodienoic acid reductase, MaOPR1, involved in the linoleic acid metabolism pathway, and functionally validated its role via dsRNA-mediated gene silencing. Results: Silencing of MaOPR1 alleviated banana leaf-lesion response after Fusarium wilt. Conclusions: Integrating metabolomic and dsRNA-silencing evidence, MaOPR1 acts as a negative regulator of banana Fusarium wilt resistance, associated with alpha-linolenic acid metabolic reprogramming in the MpICE1-overexpressing background.