Sepsis has been recognized to be a life-threatening organ dysfunction caused by the dysregulation of the host response to infections. Our work aims to screen key biomarkers related to neutrophils in sepsis using bioinformatics analysis. For this purpose, the microarray datasets related to neutrophils in sepsis patients were downloaded from the Gene Expression Omnibus (GEO) database. According to the Bayesian test, the Limma package in R was used to screen differentially expressed genes (DEGs). Then, DEGs were uploaded to the DAVID online diagnostic tool for subsequent Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment on the selected DEGs. Next, protein-protein interaction (PPI) network was established based on the selected DEGs using the STRING website and the Cytoscape software. Furthermore, according to the function of the iRegulon plug-in in Cytoscape, our study further predicts and established regulatory networks related to transcription factors and regulatory genes. In addition, the miRWalk2.0 database was used to search for miRNA-DEG pairs, associated with the conduction of intersections of miRNAs predicted by TargetScan, Miranda, miRDB and RNA22 databases. Then, these miRNA-DEG pairs were also displayed in the form of a regulatory network through Cytoscape. Finally, two datasets were selected to verify the screened genes, regulatory factors, and miRNAs, to plot receiver operating characteristics (ROC) curves and compute the area under the curve (AUC) values. The results showed that AKT1, MMP9, ARG1, ETS1 targeting AKT1, and has-miR-124-3p targeting RPS6KA5 may have diagnostic value for patients with sepsis and septic shock. While further experimental studies are required to confirm their role in septic neutrophils.