<p>Sepsis, a systemic inflammatory response syndrome (SIRS) caused by various bacterial infections, is still a severe health problem worldwide with high morbidity and mortality, whereas early diagnosis and differential diagnosis are still challenge. Human neutrophil CD64 (nCD64), also known as FcγRI, is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily that bind to the Fc (constant) region of specific portion of antibody with high affinity, which CD64 is a new specific and sensitive biomarker for early diagnosis and prognosis of sepsis. This study is aimed at selecting aptamers against human CD64 with high affinity and specificityby, a novel in vitro selection technique, termed systematic evolution of ligands by exponential enrichment (SELEX) and unraveling the molecular mechanisms of aptamer–target interactions. Human CD64 immobilized on carboxytic acid magnetic beads was subjected as the target for eight rounds of selection by SELEX technique. Bioinformatics analysis was applied for predicted secondary structures and family classification of aptamers, as well as KD values determination, and thus to determine key aptamers. Then molecular docking simulations and molecular dynamics simulations were conducted to unravel the molecular mechanisms of aptamer–target interactions. Three key aptamers were obtained after eight rounds of selection through SELEX and PCR optimization. Bioinformatics analysis showed that stem-loop and G-quartet are the major predicted secondary structures of aptamers to CD64, suggesting the potential binding sites between aptamers and CD64, and G-T mismatch is common. Using deep learning-based structure prediction, we obtained reliable three-dimensional models of the aptamers, revealing distinct structural motifs such as stem-loops and internal loops. The docking results demonstrated favorable binding conformations, with the shape complement between aptamers and electrostatic potential of the CD64 binding site. The simulations revealed the convergence and stability of the complexes, as evidenced by the relatively low root-mean-square deviation values and fluctuations. The consistency between the docking poses and the molecular dynamics results reinforces the reliability of the predicted aptamer-CD64 interactions. After eight rounds of selection, three key aptamers were obtained. Our findings provide valuable insights into the structural features and binding characteristics of the three key aptamers, supporting their potential as diagnostic reagents of CD64. This study lays the foundation for further experimental validation and optimization of these aptamers in the development of diagnostic reagents.</p>