Calcium is a necessary biochemical messenger in neuromuscular transmission, muscle compression, and hormonal secretion processes. Calcium binds with a multifunctional intermediate calcium-binding sensor protein calmodulin with a significantly higher affinity. This presented work introduces an electrochemical biosensor for the detection of sepsis by utilizing calmodulin protein on the sensing interface. The sensing platform is assembled on the gold screen-printed electrode (SPE) modified with platinum nanoparticles (PtNPs) and functionalized with chitosan. Furthermore, the calmodulin protein was electrodeposited on the chitosan functionalized SPE. The electrochemical measurements were recorded for the rapid screening of Ca2+ ions in various samples including bacterial cells and real serum samples. The serum ionized calcium is a biomarker for the detection of sepsis in newborns. The developed biosensor exhibits high sensitivity, low-cost development, and rapid results. The detection limit and sensitivity of the sensor were obtained as 1 nM and 0.51 µA/µM respectively.