Objective To investigate the distribution characteristics and drug resistance of isolates from blood culture, and to provide important reference for the diagnosis and treatment of bloodstream infection and infection control in clinical practice. Methods Positive blood culture samples of a hospital from 2016 to 2020 were collected and detected by bacteria identification and drug sensitivity analysis system. The distribution characteristics of pathogenic bacteria and drug sensitivity results were analyzed by WHONET 5.6 software. Results A total of 3 424 non-repeating strains were isolated from positive blood culture specimens, which were mainly from geriatrics department, critical care unit, emergency observation room, etc.Among them, 1 873 strains of Gram-negative bacteria were found, including Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. In recent five years, the extended-spectrum beta-lactamases Gram-negative drug resistant bacteria was slowly increasing, while other drug resistant bacteria showed little change. The overall drug resistance rates of Gram-negative bacteria to cefoperazone/sulbactam, piperacillin/tazobactam, ceftazidime, cefepime, gentamicin, tobramycin and amicacin were all less than 30%. There were 1 328 Gram-positive strains, mainly Staphylococcus, showed no resistance to datoromycin and tegacycline, and the resistance rates to linezolid, vancomycin and teicolanin were at a low level, while the resistance rates to cotrimoxazole and clindamycin showed a slow declining trend in recent five years. Conclusion Enterobacteria and Staphylococcus were the most common isolates of bloodstream infection. In clinical practice, attention should be paid to the early blood culture and drug sensitivity results, and the antimicrobial drugs should be used scientifically and rationally.