Fusarium wilt disease poses a serious threat to the global production of bananas. The targeted delivery of fungicides to banana phloem tissues may offer new hope for controlling this hard-to-treat vascular disease. In this study, fludioxonil (FLU)-loaded glycine methyl ester-conjugated polysuccinimide nanoparticles (PGA) were prepared with a loading efficiency (LE) of 27.9%. The obtained nanoparticles (FLU@PGA) exhibited pH-sensitive controlled release, specifically under an alkaline pH in plant phloem. In vivo experiments in potted bananas demonstrated that FLU@PGA can achieve the downward delivery of FLU to banana rhizomes and roots after foliar application, reducing disease severity by 50.4%. The phloem transport studies showed that the phloem loading of FLU@PGA was involved in an active transport mechanism at the organ level (castor bean seedlings). The observation of fluorescein-5-isothiocyanate cadaverine-labeled PGA nanocarriers showed that they could be absorbed by mesophyll cells and loaded into vascular tissues through the symplastic pathway. Furthermore, the interaction of FLU@PGA with the plant amino acid transporter AtLHT1 was observed to enhance transmembrane uptake at the cellular level (Xenopus oocytes). These results suggested that the phloem-targeted delivery of fungicide by transporter-mediated nanocarriers could be a promising new strategy for the management of Fusarium wilt in bananas.