Background <p>To investigate the relationship between the expression of serum PTEN and TLR4 and the severity and prognosis of neonatal septicemia and the guiding significance of management strategy.</p> Methods <p>116 newborns with sepsis admitted to the neonatal department of our hospital from March 2021 to June 2023 were recruited as research subjects. According to the Sequential Organ Failure Assessment (SOFA) scoring system, newborns with scores of 1 to 18 were set into the mild sepsis group (<i>n</i> = 64), newborns with scores from 19 to 24 were set into the severe sepsis group (<i>n</i> = 52), and 8 patients died in the severe sepsis group (death group), 44 cases survived (survival group). During the same period, 50 newborns with normal physical examination were selected as the control group. All patient guardians signed informed consent before inclusion in the study. The inflammation level of the newborns was detected by enzyme-linked immunosorbent assay and automated blood cell counter. The serum Phosphatase and Tensin Homolog (PTEN) and Toll-like Receptor 4 (TLR4) levels of the newborns were tested by enzyme-linked immunosorbent assay. The serum PTEN and TLR4 levels of newborns with severe sepsis were compared between the death group and the survival group. The correlation between PTEN, TLR4 and inflammatory indicators Procalcitonin (PCT), C-Reactive Protein (CRP), Heparin-Binding Protein (HBP), and White Blood Cell Count (WBC) was analyzed by Pearson correlation. The value of biomarkers on the condition and prognosis of newborns was analyzed through Receiver Operating Characteristic (ROC) curves.</p> Results <p>The levels of PCT, CRP, HBP and WBC in the severe sepsis group and mild sepsis group were higher than control group (<i>p</i> &lt; 0.05). The levels of PCT, CRP, HBP and WBC in the severe sepsis group were higher than mild sepsis group (<i>p</i> &lt; 0.05). The PTEN levels in the severe sepsis group and mild sepsis group were lower than control group (<i>p</i> &lt; 0.05), and the PTEN levels in the severe sepsis group were lower than mild sepsis group (<i>p</i> &lt; 0.05). The TLR4 levels in the severe sepsis group and mild sepsis group were higher than control group (<i>p</i> &lt; 0.05), and the TLR4 levels in the severe sepsis group were higher than mild sepsis group (<i>p</i> &lt; 0.05). The PCT, CRP, HBP and WBC levels of newborns in the death group were higher than survival group (<i>p</i> &lt; 0.05). The PTEN level in the death group was lower than survival group (<i>p</i> &lt; 0.05), and the serum TLR4 level in the death group was higher than survival group (<i>p</i> &lt; 0.05). PTEN was negatively correlated with PCT, CRP, HBP and WBC (<i>p</i> &lt; 0.05), that is, the increase in PTEN levels may be related to the reduction of inflammatory response. TLR4 is positively correlated with PCT, CRP, HBP and WBC indicators (<i>p</i> &lt; 0.05), indicating that when TLR4 levels increase, the levels of PCT, CRP, HBP and WBC also tend to increase. Compared with other biomarkers, PTEN and TLR4 showed high diagnostic accuracy, with Area Under the Curve (AUC) values of 0.846 and 0.857 respectively, indicating that PTEN and TLR4 are more effective than other indicators in distinguishing the health status and prognosis of newborns with sepsis (<i>p</i> &lt; 0.05).</p> Conclusion <p>Serum PTEN and TLR4 expression levels go hand in hand to the severity and prognosis of newborns with sepsis, and can be used as early predictors of severe sepsis and death in newborns with severe sepsis.</p>