In this article, yeast two-hybrid system (YTHS) and bimolecular fluorescence complementation (BiFC) were used to analyze the interactions of cucurbit aphid-borne yellows virus (CABYV)-encoded proteins. P0, P1, P1-2, P3, P4, and P5 were tested by YTHS in all possible pairwise combinations, and only P3/P3 interaction was detected. Results obtained by BiFC further confirmed the self-interaction of P3, and the subcellular localization of reconstituted YFP fluorescence was observed mainly in nuclei of Nicotiana benthamiana leaf epidermal cells. Domains involved in P3/P3 self-interaction were analyzed by YTHS and BiFC using deletion mutants. The results showed that R domain (residues 1-61) in the N-terminus could self-interact, and it also interacted with the S domain (residues 62-199) in the C-terminus of P3. The present work would serve as a molecular basis for further characterization of CABYV proteins, and the regions involved in P3/P3 self-interaction could provide the clue for understanding the capsid assembly pathway of CABYV.