The present invention relates to a method for separating nanoparticles and analyzing a biological substance using a microfluidic chip. The microfluidic chip of the present invention is effective in more sensitively and precisely detecting an analyte. According to the present invention, the use of the microfluidic chip enables the separation of nanoparticles through separation holes on the basis of size, achieving highly reliable analysis of a biological substance. In conclusion, the microfluidic chip of the present invention uses separation holes adapted to the size of nanoparticles to greatly increase the reliability of analysis of a biological substance, which will contribute to a marked improvement in the reliability of analysis based on microfluidics and a microfluidic system.