The fungus, Fusarium oxysporum f. sp. cubense (Foc), is the causal agent of Fusarium wilt disease, which is the most serious disease affecting the whole banana industry. Although extensive studies have characterized many Foc-responsive genes in banana, the molecular mechanisms on microRNA level underlying both banana defense and Foc pathogenesis are not yet fully understood. In this study, we aimed to reveal the role of miRNA during banana-Foc TR4 interactions. Illumina sequencing was used to reveal the changes in small RNAome profiles in roots of Foc TR4-inoculated 'Tianbaojiao' banana (Musa acuminata cv. Tianbaojiao) in the early stages (i.e. 5 h, 10 h and 25 h post Foc TR4 inoculation, respectively). The expression of some differentially expressed (DE) miRNAs and their predicted target genes was studied by using quantitative real time PCR (qRT-PCR). Totally, 254 known miRNAs from 31 miRNA families and 28 novel miRNAs were identified. Differential expression analysis identified 84, 77 and 74 DE miRNAs at the three respective Foc TR4 infection time points compared with control healthy banana (CK). GO and KEGG analysis revealed that most of the predicted target genes of DE miRNAs (DET) were implicated in peroxisome, fatty acid metabolism, auxin-activated signaling pathway, sulfur metabolism, lignin metabolism and so on, and many known stress responsive genes were identified to be DETs. Moreover, expected inverse correlations were confirmed between some miRNA and their corresponding target genes by using qRT-PCR analysis. Our study revealed that miRNA play important regulatory roles during the banana-Foc TR4 interaction by regulating peroxidase, fatty acid metabolism, auxin signaling, sulfur metabolism, lignin metabolism related genes and many known stress responsive genes.