This study aims to investigate the species distribution and drug resistance of pathogenic bacteria in the children with bloodstream infections in Shanghai Children’s Hospital from 2016 to 2018, and to provide rational drug use basis for clinical prevention and treatment of bloodstream infections. The automatic microbiological analysis system was used to identify the pathogenic bacteria isolated from the blood culture samples and conduct drug sensitivity test to analyze their distribution characteristics and drug resistance. A total of 24 687 samples were sent for examinations, finally 2 092 pathogenic bacteria were isolated with a positive detection rate of 8.5%. Gram-positive bacteria accounted for 84.4%, and Gram-negative bacteria accounted for 15.6%. The top five pathogens were coagulase-negative Staphylococcus (CoNS), Enterococcus, Klebsiella pneumoniae, Streptococcus and Escherichia coli. The positive detection rate of blood culture in Neonatal Department was 34.5%, and the isolation rate of CoNS was significantly higher than that in other departments. The average time to positivity of CoNS was (32.53±12.36) h. No Staphylococcus and Enterococcus resistant to vancomycin and linezolid were found. Imipenem, meropenem, amikacin, cefoperazone-sulbactam and piperacillin-tazobactam had strong antibacterial activity against Escherichia coli, and the drug resistance rate was less than 10%. The drug resistance rate of Klebsiella pneumoniae to third-generation cephalosporins reached more than 80%, and the drug resistance rate to carbapenems increased year by year, reaching 62.5% in 2018. The drug resistance rate of Pseudomonas aeruginosa to carbapenems was 9.1%. The drug resistance rate of Acinetobacter baumannii to most antimicrobial agents was more than 60%, and to carbapenems was 66.7%. It is shown that pathogenic bacteria from blood culture of children with bloodstream infections are complex, and CoNS accounts for a large proportion, which makes the diagnosis of bloodstream infections become more complicated. Therefore, understanding the distribution characteristics and drug resistance of pathogenic bacteria from blood culture of children with bloodstream infections is helpful to guide the rational use of antibiotics in clinical application.