The comprehensive identification of A3A signature sequences and preference for loop structures will enable a more accurate evaluation of A3 activity based on sequence analysis. Previous studies used only a single identified A3 signature sequence to implicate A3’s role in viral restriction or cancer progression. In contrast, our study suggests a more accurate method for determining evidence of A3 activity would be to use a set of sequences. In the case of A3A, we have identified four almost equivalent substrate signature sequences, TTCA, TTCG, CTCA, and CTCG, which should be used for identifying A3A’s involvement in mutagenesis. We also found a positive correlation between A3A’s sequence preference of binding and enzymatic activity. This correlation not only legitimizes the use of a DNA binding assay with inactive enzyme as a reliable method for studying specificity of A3s, it also shows that affinity for substrate is a driving factor for catalysis. Thus, factors that could enhance or perturb binding, such as pH or nucleic acid structure, would result in modulation of deamination activity.