Separating bacteria from infected blood is an important step in preparing samples for downstream bacteria detection and analysis. However, the extremely low bacteria concentration and extremely high blood cell count make efficient separation challenging. In this study, we introduce a method for separating bacteria from blood in a single centrifugation step, which involves sedimentation velocity-based differentiation followed by size-based cross-flow filtration over an inclined filter. Starting from 1 mL spiked whole blood, we recovered 32 ± 4% of the bacteria (Escherichia coli, Klebsiella pneumonia, or Staphylococcus aureus) within one hour while removing 99.4 ± 0.1% of the red blood cells, 98.4 ± 1.4% of the white blood cells, and 90.0 ± 2.6% of the platelets. Changing the device material could further increase bacteria recovery to >50%. We demonstrated bacterial recovery from blood spiked with 10 CFU mL⁻¹. Our simple hands-off efficient separation of low-abundant bacteria approaches clinical expectations, making the new method a promising candidate for future clinical use.