An osmotic fragility test is a useful way to determine the extent of red blood cell (RBC) hemolysis resulting from osmotic stress. A microfluidic chip-based system for measuring the osmotic fragility of RBCs has been developed. The chip was made from a Y-shaped polydimethylsiloxane (PDMS) microchannel sealed to a glass cover plate. Fresh rabbit blood diluted 1:10 with an isotonic solution and pure water respectively were introduced into the long serpentine channel using two syringe pumps. Hypotonic saline solutions with three different NaCl concentrations were prepared on a chip and images of RBCs at different locations in the channel were captured. The extent of hemolysis was estimated by comparing the cell numbers in the images using an automatic image processing program. Different degrees of hemolysis (no hemolysis, partial hemolysis, and complete hemolysis) can be estimated with this platform. This device provides a promising screening platform for diseases marked by RBC abnormalities with great simplicity, high speed and minimal requirement of blood samples.