AbstractCrohn's disease, a chronic inflammatory disorder affecting the gastrointestinal tract, is on the rise, posing significant health challenges due to its potential long‐term impact on intestinal health. Early and accurate detection of this disease is crucial for effective treatment. This study introduces an innovative approach to diagnosing Crohn's disease by utilizing a rapid and precise biosensor system as an alternative to conventional detection techniques. Utilization of an S100 A8 targeting aptamer for the specific identification of calprotectin, a well‐established biomarker associated with Crohn's disease was used as a biorecognition receptor. The aptamer is immobilized on gold nanoparticles via cysteamine and avidin to construct a biosensor. The biosensor was compared with traditional quantitative lateral‐flow tests (QLFA). The developed biosensor demonstrates that detection in 900 seconds. Performance parameters, including a limit of detection (LOD) as 5.57 μg/g and a limit of quantification (LOQ) as 16.89 μg/g, are determined through electrochemical impedance spectroscopy (EIS). Accordingly, the calibration curve equation was calculated as y=3.9736x + 1570.4 and the regression coefficient was found as R2=0.9917. These results showed that biosensor‘s capability to reliably measure calprotectin levels in real samples.