Bloodstream infections (BSIs) are a major cause of life-threatening complications in patients with cancer. According to the statistical data, the prevalence of BSIs ranges from 11% to 38%, and overall mortality reaches about 40%. Immediate antibiotic treatment could greatly shorten hospital stays, decrease the mortality rate and decrease healthcare costs. Therefore, a fast detection of bacteria for BSI determination is in high demand. Here, a novel paper-based microfluidic chip with water-absorbing material for generating sample fluid flow is designed for the detection of bacteria in whole blood. The corresponding green quantum dots for the labelling of target S. aureus are prepared by modifying their surfaces with an aptamer for specific binding. Blood cells are separated during diluted sample solution flow in the chip to eliminate interferences and the labelled bacteria are captured at the detection area in the chip. Fluorescent intensity is then measured using a microscope to determine the number of target bacteria in whole blood. The assay of S. aureus in whole blood, which is prepared by adding bacteria into the blood sample, shows that this method could detect bacteria in less than 20 min with a detection limit as low as 10 CFU/mL. It is much faster than clinical tests using the culture method, PCR or mass spectroscopy. The developed method does not require the use of large-scale equipment and complex manual operations. This point-of-care-testing (POCT) method is envisioned to be used for the rapid assessment of certain bacterial infection in BSIs.