The polysaccharide cell wall component, 1,3-beta-D-glucan (BDG), is used as a serum biomarker for invasive fungal infection (IFI). Patients receiving hematopoietic stem cell transplantation (HSCT) are considered a highly vulnerable group for IFI development. We evaluated the diagnostic performance of serum BDG screening in HSCT recipients. HSCT recipients were prospectively enrolled in this study between September 2014 and August 2015. Routine serum BDG screening was performed 2-3 times weekly by using the Fungitell(®) assay. All samples were classified according to the 2008 EORTC/MSG criteria, with serum BDG results not being considered for classification. The diagnostic performance of BDG testing for IFI was calculated. BDG values ≥80 pg/mL were considered positive. A total of 308 serum samples were collected in 45 patients. The majority of 172 samples (55.8%) were obtained at the early phase (within 30 days) after allogeneic HSCT. BDG levels were significantly higher in 16 possible/probable IFI samples when compared to no evidence for IFI samples (median 170 pg/mL, interquartile range [IQR] 100-274 pg/mL vs. median 15 pg/mL, IQR 15-15 pg/mL; P < 0.001, Mann-Whitney U-test). Diagnostic performance of serum BDG screening for possible IFI/probable invasive pulmonary aspergillosis vs. no evidence for IFI was as follows: sensitivity 81%, specificity 98%, positive predictive value 65%, negative predictive value (NPV) 99%, and diagnostic odds ratio 176 (95% confidence interval 41-761). Our data suggest that serum BDG testing in HSCT patients may be highly specific and associated with a very high NPV of >99%. Therefore, serum BDG may be a helpful tool to rule out IFI in HSCT patients.