Provided are a microfluidic chip for multiple bioassay and a method of manufacturing the same. The method includes: forming microfluidic channels by coupling a channel structure having grooves for sample channels and probe channels in a bottom surface and a substrate; forming probe immobilization regions at intersections between the probe channels and the sample channels; and forming blocking walls in the probe channels before and after each of the probe immobilization regions to prevent mixing of target sample. Since the probes are immobilized in the microfluidic channels after the formation of the microfluidic channels, difficulty in connecting fluidic channels after the immobilization of probes on a substrate can be removed. In addition, a microfluidic platform for a multiple bioassay into which a plurality of samples can be simultaneously loaded can be formed.