Sepsis is a potentially fatal systemic body infection with a significant mortality rate worldwide. Procalcitonin (PCT) is a sensitive and specific biomarker for sepsis diagnosis, however, the traditional immune-gold labeling detection of PCT is semi-quantitative with relatively low sensitivity (>0.1 ng mL⁻¹). Recently proposed techniques (such as electrochemical immunosensor, microarray, and total internal reflection based methods) require either a complex sensor or sophisticated equipment. Herein, we report a magnetic bead and enzyme-antibody labeled gold nanoparticle based ELISA strategy for sensitive PCT detection. This developed assay is sensitive enough to detect PCT at 20 pg mL⁻¹, efficient for the direct human serum test. Notably, this assay specifically distinguishes PCT from other sepsis markers and the whole assay takes only 1.5 hours to finish.