A detailed elucidation of the factors affecting the binding of anti-double-stranded (ds)-DNA antibody to ds-DNA immobilized on surfaces may provide insight into the pathogenicity of the systemic lupus erythematosus disease and lead to the development of surfaces for specific DNA and antibody recognition. In this work, the influence of the surface parameters on the attachment of antibody to DNA under physiologically relevant conditions was studied by magnetic alternating current mode atomic force microscopy, ellipsometry, and fluorescence spectroscopy. The DNA surface orientation (tethered vs flat lying), duplex surface density, antibody concentration, and reaction time were all found to affect the antibody/DNA conjugate formation. The mouse monoclonal antibody was specific toward ds-DNA over single-stranded DNA. The steric hindrance and reduced flexibility of surface-confined polynucleotide DNA molecules for the conjugate formation were examined by comparing the conjugates attached to calf thymus DNA preimmobilized onto a surface to those preformed in a homogeneous solution comprising DNA and antibody molecules and subsequently attached to the surface. Antibody aggregation was also found to be prevalent at surface sites where DNA/antibody conjugates existed.