Objectives: Biomarkers are widely used for rapid diagnosis of sepsis. This study evaluated the diagnostic accuracy of presepsin, procalcitonin (PCT), and C-reactive protein (CRP) in differentiating sepsis severity as well as their association with Sepsis-related Organ Failure Assessment (SOFA) score. ----- Methods: One hundred septic patients from two university clinical centers were enrolled in the study during two time periods. New Sepsis-3 definitions were used for sepsis stratification. Biomarkers and SOFA score were evaluated four times during the illness. A sandwich ELISA kit was used for presepsin measurement. Generalized linear mixed effects model was used to test the changes in biomarkers concentrations and SOFA score values during the illness and to estimate the differences between severity groups. Multivariate analysis was used to test the association of biomarkers with SOFA score. ----- Results: Presepsin concentrations were significantly higher on admission in patients with septic shock (n = 34) compared to patients with sepsis (n = 66), mean ± SD: 128.5 ± 47.6 ng/mL vs. 88.6 ± 65.6 ng/mL, respectively (p < 0.001). The same was not observed for PCT and CRP; their concentrations did not differ significantly between severity groups. A strong correlation of presepsin with SOFA score was also found (p < 0.0001). ----- Conclusions: Presepsin had a good diagnostic ability to differentiate septic shock from sepsis in the study groups. PCT and CRP failed in differentiating sepsis severity.