The separation or purification of bacterial samples from a mixed cell suspension is critical in a variety of biomedical applications, such as sputum diagnostics and cell biology studies. We propose a streamline-based microfluidic filtration device for highly efficient purification of bacterial samples from a mixed cell suspension. The device is composed of tens of repeated streamline-based separation units that continuously filter the solution. By injecting a liquid sample such as liquefied human sputum solution through the device, approximately 50% of the injected sample solution can be collected from the filtration collection channels, which filter approximately 99.9% of the mammalian cells but retain approximately 60% to 90% of the bacteria. Different injection rates (0.2 ml h-1 to 30 ml h-1), different sample viscosities, and different initial bacterial densities were tested and confirmed that our separation method was robust. The easy operation, robustness and high efficiency indicate that our method may be useful for the separation or purification of bacterial samples from a mixed cell suspension, such as bacterial samples for sputum diagnostics.