As the largest family among the plant resistance (R) proteins, the nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins play significant roles in the defense of pathogens. The completion and improvement of banana reference sequence make a systematic insight into the banana NBS-LRR protein family possible. In this study, a total of 98 NBS-LRR proteins were identified from the banana genome and clustered into eight classes in the phylogenetic tree. NBS-LRR genes were unevenly distributed on all 11 chromosomes of banana. Typical NB-ARC (nucleotide binding-APAF-1, disease resistance proteins, CED-4) and LRR motifs were found in all NBS-LRR proteins and a small number of introns existed in most NBS-LRR genes. Transcriptional profiles of NBS-LRRs were investigated in Fusarium oxysporum f. sp. cubense (Foc) susceptible and resistant banana cultivars BX and HDJ challenged with Foc race 1 (Foc 1) and tropical race 4 (Foc TR4). Among 31 representative NBS-LRRs detected in this study, 12 members showed stronger transcriptional stimulation in HDJ than in BX, while in contrast, eight members exhibited higher expression in BX than in HDJ. This study advances our understanding of roles of banana NBS-LRRs in the defense of Fusarium wilt and will contribute to the genetic improvement of banana resistance.