Residues 221-239 of rubella virus E1 glycoprotein contain antibody neutralization domains, and the solvent-exposed charged amino acids at the binding interface may be crucial for binding ability. However, the role of charged amino acid residues on the E1 epitope in peptide-antibody binding is unknown. To investigate the role of single amino acid substitutions on the important neutralizing epitope, biolayer interferometry and serological tests were performed. There are three charged residues in the neutralizing epitope: D229, R237, and H238. Substitution of D229 for amino acid A had no influence on the binding activity of the antibody to the peptide. However, substitutions of R237 or H238 for charged amino acid H or R were found to abolish the binding activity. Furthermore, substitution of an uncharged amino acid Q236 for a charged amino acid D was found to reduce the binding activity significantly. Thus, R237 and H238 are key amino acids in the rubella virus E1 neutralization epitope.