<p>Myeloperoxidase (MPO) plays a crucial role in physiological regulation and pathological processes. Fluctuations in MPO levels are recognized as significant biomarkers for early disease detection and precision diagnostics. Current biochemical technologies face limitations in the quantitative analysis of MPO, including insufficient sensitivity, labor-intensive procedures, and high costs, which constrain its broader application. DNA aptamers, a class of functional nucleic acid, are capable of recognizing and binding to targets. They are widely utilized in the development of biosensing and therapeutic applications. However, research on MPO-specific aptamers remains limited. Traditional screening methods, such as systematic evolution of ligands by exponential enrichment, suffer from inefficiencies, high costs, and challenges in optimizing aptamer performance. This study leveraged a one-step hydrogel-based screening method inspired by hydrogel technology. This innovative approach not only simplified the aptamer screening and improved efficiency but also enabled the capture of high-affinity MPO aptamers. Furthermore, the binding affinity of these aptamers to MPO was quantified by determining their affinity constants, validating their strong binding capabilities. As a proof-of-concept application, a magnetic separation system was designed to facilitate MPO-specific detection using nucleic acid sensors. This work establishes a solid foundation for the application of MPO aptamers in diagnostics and other related fields.</p>