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Nucleic acid aptamers have shown a broad application prospect in basic research, clinical diagnosis and treatment, new drug development and various other fields. We have screened the DNA aptamer A1 and A2 target at Cε3-Cε4 with high affinity and specificity, another aptamer A8, no affinity with Cε3-Cε4 protein, was as a negative control in this study. The structures of aptamer A1 and A2 were optimized using the deletion method, complementary sequence method, and point mutation method, to make them perform biological functions better, improve the pertinence of the subsequent modification and study the mechanism of action of aptamers coupled Cε3-Cε4. Additionally, the affinity was detected using competitive ELISA, then the most optimal and minimalist aptamer G39-A1-29C was obtained. The results indicated that the G39-A1-29C can significantly inhibit the IgE-dependent cell degranulation, but no effect in IgE-independent manner, and have a notable therapeutic effect with dose-dependent on PCA experiments in vivo. Moreover, it is found that the aptamer maintains the secondary structure through the fixed sequence, consecutive four GC pairings can significantly increase the binding affinity, and the G base on the loop region of A1 may be the key sites for binding to the domain of the target protein Cε3-Cε4. Therefore, the stem-loop structure of A1 is the structural basis of its binding, too short sequence cannot maintain the secondary structure, so that its affinity is significantly reduced. The results facilitated the modification and chemical synthesis of aptamers in next work, which provided the foundation for the development of new drugs for the treatment of allergy diseases.