Oleosin is the most abundant protein on the surface of oil body, which plays an important role in the formation of plant oil body and oil storage. Cyperus esculentus is a unique tuber oil crop. Exploring the characteristics of the oleosin gene family in Cyperus esculentus and screening the key oleosin genes responding to oil storage are of great significance for analyzing the regulation mechanism of oil storage. In this study, the phylogenetic and collinear analysis of oleosin gene family members in Cyperus esculentus and several other species were performed using bioinformatics methods, and further tissue expression analysis of CeOLEs, oil content detection in different tissues, and functional verification of overexpressing CeOLEs in Arabidopsis were carried out. Bioinformatics analysis showed that six oleosin gene family members (CeOLE1 - 6) were identified in the whole genome of Cyperus esculentus, the number of which was relatively small; however, they distributed in three evolutionary lineages (U, SL and SH subfamilies), and have high evolutionary conservation and collinearity with oleosin gene family members of maize and rice. The qPCR analysis showed that the expression levels of the six CeOLEs were high in the tubers of Cyperus esculentus, but extremely low in roots, leaves, tillering nodes, stolons and other tissues. Accordingly, the oil content of Cyperus esculentus tubers was up to 24.68%, while the oil content in roots, leaves, tillering nodes and stolons was less than 1.00%. It can be seen that the oil content of Cyperus esculentus may be closely positively correlated with the expression level of CeOLEs. In addition, through the analysis of the expression level of CeOLEs and oil content in tubers at different development stages, it was found that the expression levels of CeOLE1, CeOLE2, CeOLE3 and CeOLE5 were higher, and their expression patterns were consistent with the change rate of oil content in tubers (first increased and then decreased), indicating that these four CeOLEs may be the key genes affecting the oil content in Cyperus esculentus tubers. Overexpression of CeOLE1, CeOLE2, CeOLE3 and CeOLE5 in Arabidopsis significantly increased the oil content of Arabidopsis seeds, which verified the function of these four CeOLEs in oil storage. In summary, the results of this study clarified the characteristics of oleosin gene family in Cyperus esculentus, screened and functionally verified the key oleosin genes responding to oil storage, and laid a foundation for further analysis of oil storage mechanism and genetic improvement of Cyperus esculentus.