Objective To understand the clinical characteristics of bloodstream infection (BSI) of Staphylococcus aureus (SA) in children and antimicrobial susceptibility characteristics of the pathogenic strains, and explore the susceptibility factors of methicillin-resistant SA (MRSA) BSI and SA healthcare-associated infection (HAI). Methods Medical records of hospitalized children with SA isolated from blood culture in a children's hospital from January 2014 to December 2019 were retrospectively analyzed. According to antimicrobial susceptibility test results and sources of SA, patients were divided into MRSA group, methicillin-sensitive SA (MSSA) group, as well as healthcare-associated BSI group (HA group) and community-associated BSI group (CA group). Chi-quare test and logistic regression were used to analyze the risk factors for MRSA BSI and HA-BSI. Results A total of 143 cases were included, the ratio of male to female was 1.8 ∶1, 71 cases (49.6%) were < 1 year old, and 90 cases (62.9%) were with focal BSI. 50 cases (35.0%) were in MARS group and 93 cases (65.0%) in MSSA group; 73 cases (51.0%) were in HA group and 70 cases (49.0%) in CA group. Mechanical ventilation (OR=17.320, 95%CI [1.576-190.399]) and combined use of antimicrobials (OR=0.580, 95%CI [0.359-0.938]) were independent risk factors for MRSA BSI (both P < 0.05). Mechanical ventilation (OR=31.466, 95%CI [1.434-690.538]), use of antimicrobials before admission (OR=24.524, 95%CI [1.699-353.915]), length of hospital stay >7 days (OR=1.090, 95%CI [1.010-1.176]), use of antimicrobials >7 days (OR=0.910, 95%CI [0.842-0.984]) were independent risk factors for HA BSI (all P < 0.05). Resistance rates of MRSA to erythromycin, clindamycin and rifampicin were 82.0%, 76.0% and 32.0% respectively, all were higher than those of MSSA (all P < 0.05). 6 cases (8.2%) in HA group and 4 cases (5.7%) in CA group died/not cured, with no statistically significant diffe-rence between two groups (P > 0.05). Intravenous catheterization, mechanical ventilation and admission to ICU were risk factors for poor prognosis of children with SA BSI (all P < 0.05). Conclusion The majority of children with SA BSI are male, mainly at the age of < 1 year old, and focal BSI is more common. Mechanical ventilation and combined use of antimicrobials are risk factor for MRSA infection. HA BSI often occurs in patients with mechanical ventilation, use of antimicrobials before admission, length of hospital stay >7 days, and use of antimicrobials >7 days. Targeted treatment as well as prevention and control measures should be taken according to antimicrobial susceptibility test results and infection types.