Background Newborns are particularly susceptible to infection in hospitals, with neonatal sepsis being the most common infection symptom and the third leading cause of neonatal death. Klebsiella pneumoniae is a gram-negative bacterium of Enterobacteriaceae, which is a common pathogen of neonatal septicemia. In this study, we will analyze and evaluate the current status, clinical characteristics, and drug resistance of Klebsiella pneumoniaesepsis infection in Neonatal Intensive Care Unit (NICU), with the aim of providing effective basis for timely and accurate clinical diagnosis and treatment in clinical practice. Methods Statistical analysis was performed on 75 cases of Enterobacteriaceae septicemia in infants admitted to NICU in a special obstetrics and gynecology hospital in Shanghai from January 2020 to June 2022. Based on bacterial identification, isolates were divided into the Klebsiella pneumoniae (KP) group (n = 49) and the non-KP Enterobacteriaceae group (n = 26). The infection, clinical characteristics, and bacterial resistance of the two groups of infected patients were compared. Results Comparing the clinical characteristics of the two groups, the results showed that most of the subjects in the KP and non-KP groups were premature infants, accounting for 100% and 92.3% of subjects, respectively; late onset was the main disease in both groups, accounting for 93.9% and 80.8% of subjects, respectively. All patients received Peripherally Inserted Central Catheter(PICC). The levels of pro calcitonin and CRP (C-reactive protein) were significantly higher in the KP group compared with those in the non-KP group (p < .05). At the same time, the incidence of thrombocytopenia in the KP group was significantly higher than that in the non-KP group (p < .05). The proportion of antimicrobial drug exposure in the KP group is higher than that in the non-KP group. The drug resistance of the KP group to ceftazidime, ceftriaxone, cefepime, ampicillin/sulbactam, aztreonam, ciprofloxacin and compound sulfamethoxazole was significantly higher than that of the non-KP group, whereas the drug resistance rate to cefotetan, gentamycin and to bramycin was significantly lower than that of the non-KP group, Statistically significant differences (p < .05). 38 cases of Klebsiella pneumoniae producing ESBLs were tested for related resistance genes. The results showed that the main resistance types were SHV and TEM, with detection rates of 60.6% and 28.9%. Conclusions This study shows that neonatal sepsis caused by Klebsiella pneumoniae infection has a high incidence and drug resistance in premature and low birth weight infants, and has become a serious public health problem; Clinicians should pay attention to differential diagnosis, Reasonable selection of antibiotics to reduce the generation of drug-resistant bacteria.