Aim. This study aimed to synthesize, purify, and investigate the interactions between oligonucleotides (OLN) and recombinant signaling proteins, specifically interferon α2-β, insulin, their receptors, and somatropin, using docking and fluorescence spectroscopy methods. Methods. To analyze these interactions, we employed the Stern-Volmer equation in its general and modified forms, as well as the Gill equation, which enabled us to determine the binding and binding constants. We synthesized oligonucleotides using the solid-phase phosphoamidite method, which offers high efficiency and specificity. The resulting oligonucleotides were then purified by solid-phase extraction, which removed by-products and impurities, as confirmed by spectral analysis. Results. Fluorometric titration revealed that homopolymeric oligonucleotides bind to proteins within the medium-affinity range, forming non-fluorescent complexes. The most significant interactions occurred with shorter oligonucleotides. We observed negative cooperative binding between insulin and the oligoribonucleotide Poly(rG)₅. Notably, all proteins selectively bound only one ligand, the oligonucleotide Poly(dG)₅. Conclusions. Understanding the mechanisms of protein–oligonucleotide interactions may open new possibilities for developing antibiotics, antiviral drugs, and treatments for cancer and genetic diseases.