The exon junction complex (EJC) plays important roles in RNA metabolisms and the development of eukaryotic organisms. MAGO (short form of MAGO NASHI) and Y14 (also Tsunagi or RBM8) are the EJC core components. Their biological roles have been well investigated in various species, but the evolutionary patterns of the two gene families and their protein-protein interactions are poorly known. Genome-wide survey suggested that the MAGO and Y14 two gene families originated in eukaryotic organisms with the maintenance of a low copy. We found that the two protein families evolved slowly; however, the MAGO family under stringent purifying selection evolved more slowly than the Y14 family that was under relative relaxed purifying selection. MAGO and Y14 were obliged to form heterodimer in a eukaryotic organism, and this obligate mode was plesiomorphic. Lack of binding of MAGO to Y14 as functional barrier was observed only among distantly species, suggesting that a slow co-evolution of the two protein families. Inter-protein co-evolutionary signal was further quantified in analyses of the Tol-MirroTree and co-evolution analysis using protein sequences. About 20% of the 41 significantly correlated mutation groups (involving 97 residues) predicted between the two families was clade-specific. Moreover, around half of the predicted co-evolved groups and nearly all clade-specific residues fell into the minimal interaction domains of the two protein families. The mutagenesis effects of the clade-specific residues strengthened that the co-evolution is required for obligate MAGO-Y14 heterodimerization mode. In turn, the obliged heterodimerization in an organism serves as a strong functional constraint for the co-evolution of the MAGO and Y14 families. Such a co-evolution allows maintaining the interaction between the proteins through large evolutionary time scales. Our work shed a light on functional evolution of the EJC genes in eukaryotes, and facilitates to understand the co-evolutionary processes among protein families.