Brochosomes are a unique coating on the integuments of Cicadellidae produced in specialized glandular sections of Malpighian tubules. However, knowledge of the protein composition of brochosome is very limited. In this study, transcriptomic and proteomic profiling was performed to characterize the protein composition of the brochosome in rice green leafhopper Nephotettix cincticeps. Brochosomes were collected from the forewings of leafhoppers using ultrasonic treatment allowing for more effective brochosome collection and shock treatment resulting in purer brochosomes . After combined proteomic and transcriptomic analysis, 12 putative major structural proteins of the brochosome (BSM) and 10 brochosome-associated proteins (BSAP) were identified. Each BSM has specific conserved motifs containing several cysteines cross-linked by disulfide bonds. BSAP, which may be loosely linked to the outer and inner surfaces of the brochosomes, might play an important role in the proper folding of the BSM and resisting pathogens. Apart from a few BSAPs associated with cyclase, homologs of BSM and BSAP were present exclusively in the insect family Cicadellidae rather than other Hemiptera species, suggesting that these proteins might be encoded by an orphan gene family specific to the Cicadellidae. This is the first study to identify and characterize the protein composition of the brochosome of N. cincticeps, which will provide a solid foundation for further studies of brochosome synthesis, function, and evolutionary origins.