One of the largest and most significant transcription factor gene families in plants is the SQUAMOSA promoter binding protein (SBP) gene family and they perform critical regulatory roles in floral enhancement, fruit development, and stress resistance. The SBP protein family (also known as SPL) has not yet been thoroughly studied in the staple fruit crop, banana. A perennial monocot plant, banana is essential for ensuring food and nutrition security. This work detected 41 SBP genes in the banana species Musa acuminata. The MaSBPs were subsequently elucidated by investigating their gene structure, chromosomal position, RNA-Seq data, along with evolutionary connections with Arabidopsis and rice. Sequence alignment of MaSBPs revealed that all genes included a domain of two Zn finger motifs (CCCH and CCHC motifs) with an overlapping nuclear localization signal region. The conserved motifs sequence in the inferred MaSBP proteins were quite comparable. According to findings, the time frame of divergence for duplicated MaSBP gene pairs ranged from 42.39 to 109.11 million years and the dicot Arabidopsis and monocotyledonous plant banana diverged before the division of banana and monocot rice. Moreover, cis-acting element and GO annotation analysis exhibited possible biological activities of MaSBPs in flower development, phytohormone regulation, and stress tolerance. RNA-Seq expression profiling exhibited that genes MaSBP-3, MaSBP-20, MaSBP-37, MaSBP-40 were more expressed during floral and fruit development stage. The foundation for additional investigation of SBP protein sequences in other plants can be laid out by this study, which will shed light on some of their crucial biological functions.