Introduction: Red blood cell (RBC) transfusion is associated with poor clinical outcome in critically ill patients. We investigated the predictive value of biomarkers on intensive care units (ICU) admission for RBC transfusion within 28 days. Methods: Critically ill patients (n = 175) who admitted to our ICU with organ dysfunction and an expected stay of ≥ 48 hours, without hemorrhage, were prospectively studied (derivation cohort, n = 121; validation cohort, n = 54). Serum levels of 12 biomarkers (hemoglobin, creatinine, albumin, interleukin-6 [IL-6], erythropoietin, Fe, total iron binding capacity [TIBC], transferrin, ferritin, transferrin saturation, folate, and vitamin B12) were measured upon ICU admission, days 7, 14, 21 and 28. Results: Among the 12 biomarkers measured upon ICU admission, levels of hemoglobin, albumin, IL-6, TIBC, transferrin and ferritin were statistically different between transfusion and non-transfusion group. Of 6 biomarkers, TIBC upon ICU admission had the highest area under the curve value (0.835 [95% confidence interval] = 0.765–0.906) for predicting RBC transfusion (cut-off value = 234.5 µg/dL; sensitivity = 0.906, specificity = 0.632). This result was confirmed in validation cohort, whose sensitivity and specificity were 0.888 and 0.694, respectively. Measurement of these biomarkers every seven days revealed that albumin, TIBC and transferrin were statistically different between groups throughout hospitalization until 28 days. In validation cohort, patients in the transfusion group had significantly higher serum hepcidin levels than those in the non-transfusion group (P = 0.004). In addition, joint analysis across derivation and validation cohorts revealed that the serum IL-6 levels were higher in the transfusion group (P = 0.0014). Conclusion: Decreased TIBC upon ICU admission has high predictive value for RBC transfusion unrelated to hemorrhage within 28 days.