The study aimed to synthesize, purify, and investigate the interaction of oligonucleotides (OLNs) with recombinant signaling proteins and their receptors, assessing the binding strength using fluorescence analysis and the Stern-Volmer equation. OLN-1 to OLN-8 were synthesized and purified, and their purity confirmed by HPLC. Fluorometric titration revealed static binding of OLNs to proteins, forming non-fluorescent complexes, except for OLN-3 with insulin (INS) and OLN-2 with interferon α2-β (INF α2-β), showing mixed binding. This indicates that these proteins interacted with OLNs at very low concentrations. OLN-1, OLN-6, OLN-7, and OLN-8 exhibited high binding activity to all proteins. Moreover, positive cooperative binding (2A+B=A2B) was observed in some cases, where one OLN molecule facilitated the attachment of the next due to conformational changes. OLN-3 and OLN-4 displayed significant positive cooperative binding with INF α2-β. In conclusion, the study demonstrated a strong interaction between OLNs and recombinant signaling proteins and receptors, potentially influencing their conformation and biological activity. These findings have implications for the therapeutic use of OLNs in the context of signaling proteins and receptors.