Objective To explore the risk factors, clinical characteristics and pathogenic bacteria of late-onset septicemia (LOS) in neonates, so as to guide clinical diagnosis and treatment. Methods Collect LOS in neonates admitted to the Department of Pediatrics, Chaohu Hospital of Anhui Medical University between January 2015 and February 2020, and set them as the observation group. The neonates born at the same time and hospitalized without septicemia were selected as the control group. The general situation and risk factors of the two groups of neonates were analyzed, and the clinical manifestations, complications and pathogenic bacteria of LOS in neonates were analyzed. Results 182 neonates were enrolled, 91 in each group. There were significant differences between the two groups in mechanical ventilation, indwelling peripherally inserted central catheter, parenteral nutrition, tracheal intubation, neonatal asphyxia, gestational age<37 weeks, birth weight<2.0 kg (P<0.05). Logistic regression analysis showed that gestational age<37 weeks [odds ratio (OR)=3.010, 95% confidence interval (CI) (1.489, 6.085), P=0.002], parenteral nutrition [OR=3.506, 95%CI (1.681, 7.312), P=0.001] were independent risk factors for LOS. The main clinical manifestations of LOS were abnormal temperature, feeding difficulties, jaundice, apnea, hypersensitive C-reactive protein and procalcitonin increase. The neonates with LOS were prone to necrotizing enterocolitis and purulent meningitis. A total of 74 pathogenic bacteria were cultured from neonates with LOS, including 49 Gram-positive bacterium, 21 Gram-negative bacteria and 4 fungi. The critical and death cases were mainly infected by Gram-negative bacteria. Conclusions A number of factors are related to LOS. Gestational age<37 weeks and parenteral nutrition are independent risk factors for LOS. In order to avoid LOS, attention should be paid to prevention, aseptic concept should be strengthened, and drugs should be used reasonably.