Prior studies indicate that serum calprotectin (SC) and plasma calprotectin (PC) can be used as biomarkers in Crohn's disease (CD). The aim of this study was to investigate the diagnostic accuracy of SC and PC in patients with a clinical suspicion of CD.This biobank study included patients from a prospective, blinded, multicenter study examining minimally invasive modalities for diagnosing CD. Patients had a standardized work-up including ileocolonoscopy, pan-enteric capsule endoscopy, and blood samples within a 2-week period. Plasma and serum were stored at - 80 °C until further analysis. A routine C-reactive protein (CRP) was measured on the same day. Pan-endoscopy served as reference standard.126 patients entered the study, and 58 (46.0%) were diagnosed with CD. Patients with CD had a median PC of 0.37 mg/L (IQR 0.20-0.70) compared to 0.29 mg/L (IQR 0.16-0.41) in non-CD patients (p = 0.03). The median SC was 1.09 mg/L (IQR 0.80-1.80) and 0.93 mg/L (IQR 0.66-1.25), respectively (p = 0.01). Receiver operating characteristics curves showed an AUC of 0.63 (CI 0.53-0.73) for SC and 0.61 (CI 0.51-0.71) for PC for detection of CD, which was inferior to that of CRP (AUC = 0.76, CI 0.68-0.85) (p < 0.02). None of the biomarkers reflected the endoscopic severity of CD.Although levels of PC and SC are elevated in patients with CD, diagnostic accuracies are inferior to CRP. SC and PC are not reliable as stand-alone blood-based biomarkers for diagnosing CD and selecting patients for endoscopy.