The soil-borne fungal pathogen Fusarium oxysporum f.sp. is responsible for Fusarium wilt. cubense tropical race 4, is one of the most devastating diseases in bananas, regarded as a major yield-reducing factor in the banana industry worldwide. Understanding the molecular interactions in banana defense responses is an important tool to reveal the unexplained processes that underlie banana resistance to Fusarium oxysporum f. sp. cubense tropical race 4. The seedlings of moderately resistant variety Guijiao No. 9 and a susceptible cultivar Guijiao No. 6 were cultured in tissue culture, and the characterize protein profile expression changes responses to after inoculation the Fusarium oxysporum f. sp. of cubense tropical race 4 were detected by isobaric labeling based on MS2 quantification at the 2nd, 4th, 6th and 8th day. Interestingly, new genes in the resistance of banana to Foc37-GFP were identified, including several other serine/threonine-protein kinase, AvrRpt-cleavage domain-containing protein, peptidylprolyl isomerase and some Jacalin-type lectin domain, the resistance-related pathways “ribosome”, “microbial metabolism in diverse environments”,“carbon metabolism”,“biosynthesis of amino acids”and “biosynthesis of antibiotics” pathways were significantly enriched, the resistant banana cultivar Guijiao 9 shows formation of different constitutive cell barriers to restrict spreading of Fusarium oxysporum f. sp. cubense tropical race 4. In this study, the dynamic change root proteomic of moderately resistant cultivar Guijiao 9 and a susceptible cultivar Guijiao 6 were characterized and provided a differentially expressed proteins comparative analysis of the compatible and incompatible interaction between Fusarium oxysporum f. sp. cubense tropical race 4 and banana. These findings provide a substantial contribution to existing sequence resources for banana, and a strong basis for future proteomic research. The proteins that displayed two-fold changes in intensity are related to biochemical processes that may be differentially altered at various times after Fusarium oxysporum f. sp. cubense tropical race 4 infection. These findings will accelerate research on resistance in banana to Fusarium oxysporum f. sp. cubense tropical race 4 and contribute to a better understanding of the banana defense mechanism to plant pathogens, hopefully.