The fruitless (fru) gene is a key factor in controlling sexual behavior in insects. The homolog of fru has been identified in most insect species and exhibits conservation in the transcript architecture and regulation of male sexual behavior. However, the structure of fru transcripts in Hemiptera remains unknown. Here, we identified and characterized two fru transcripts in Bemisia tabaci, named fru-a and fru-b. fru-a contained a 1263 bp open reading frame (ORF) encoding for 420 amino acids, while fru-b contained a 1143 bp ORF encoding for 380 amino acids. These two proteins start with the same BTB domain and end with two different zinc finger domains, belonging to ZnA and ZnG, respectively. The expression of fru-a and fru-b differed significantly between females and males, and both were expressed at lower levels in males. We demonstrated the presence of multiple TRA/TRA-2 binding sites and alternative splicing in fru-a and fru-b. Moreover, the RNAi result provided evidence that transformer regulates the expression of fru-a and fru-b. These results promote the study of the sex determination cascade in B. tabaci and lay the foundation for the study of sexual behavior in this insect.