Blood stream infection is one of the major public health issues characterized with high cost and high mortality. Timely effective antibiotics usage to control infection is crucial for patients' survival. The standard microbiological diagnosis of infection however can last days. the delay in accurate antibiotic therapy would lead to not only poor clinical outcomes, but also to a rise in antibiotic resistance due to widespread use of empirical broad-spectrum antibiotics. An important measure to tackle this problem is fast determination of bacterial antibiotic susceptibility to optimize antibiotic treatment. We show that a protocol based on surface-enhanced Raman spectroscopy can obtain consistent antibiotic susceptibility test results from clinical blood-culture samples within four hours. the characteristic spectral signatures of the obtained spectra of Staphylococcus aureus and Escherichia coli-prototypic Gram-positive and Gram-negative bacteria-became prominent after an effective pretreatment procedure removed strong interferences from blood constituents. Using them as the biomarkers of bacterial metabolic responses to antibiotics, the protocol reported the susceptibility profiles of tested drugs against these two bacteria acquired from patients' blood with high specificity, sensitivity and speed. Blood stream infection (BSI)-defined as the presence of viable bacteria in blood (i.e., bacteremia) documented by a positive blood culture result 1-arouses morbidity and mortality worldwide 2. It is the primary cause of sepsis, which is a major public health concern with particular high medical cost 3,4. Although the number of BSI incidence bears demographic difference, it shows increasing trend over time 5,6 and therefore remains an urgent medical challenge. Timely administration of appropriate antibiotic therapy is critical to avoiding its progression. A retrospective analysis study based on a large dataset showed that there was a linear increase in the risk of mortality for each hour delay in antibiotic administration 7 , which was confirmed by a recent study 8. However, owing to lack of timely microbiological evidence, antibiotics are usually forced to start empirically, rather than precisely upon specific target(s) 9. Improper antibiotic selection was not uncommon and the mortality markedly increased 10. Moreover, the initial empirical use of broad-spectrum antibiotics 11 , although prudent, inevitably