Sepsis is a syndrome without a standard validated diagnostic test. Early recognition is crucial. Serum proteome analysis in children with sepsis may identify new biomarkers. This study aimed to find suitable blood biomarkers for an early diagnosis of sepsis. An analytical observational case-control study was carried out in a single center. Children admitted to a Pediatric Intensive Care Unit with clinical diagnosed sepsis were eligible for study. A proteomic analysis conducted by mass spectrometry was performed. Forty patients with sepsis and 24 healthy donors were recruited. Proteomics results revealed 44 proteins differentially expressed between patients and healthy controls. Six proteins were selected to be validated: lactoferrin, serum amyloid-A1 (SAA-1), complement factor B, leucine-rich alpha-2 glycoprotein (LRG1), soluble interleukin-2 alpha chain receptor (sCD25) and soluble haptoglobin-hemoglobin receptor. Our results showed that sCD25, SAA-1, and LRG1 had high levels of specificity and sensitivity, as well as an excellent area under the ROC curve (>0.9). Our study provides a serum proteomic analysis that identifies new diagnostic biomarkers in sepsis. SAA-1, sCD25 and LRG1 were able to separate septic from healthy donor, so they could be used together with other clinical and analytical features to improve sepsis diagnosis in children.