The invention discloses a microfluidic paper based sensor based on fluoroimmunoassay and utilizing a quantum dot as a marker to detect norfloxacin (NOR) in milk and belongs to the technical field of biological nanometer. A preparation method of the sensor comprises the steps: firstly, forming a hydrophilic area and a hydrophobic area on chromatographic paper through a wax spray printing technology, roasting for a certain time and performing plasma treatment on the paper; then, preparing NOR-bovine serum albumin (BSA) coating antigen and a quantum dot marked NOR antibody; then, respectively adding the coating antigen and a goat anti-mouse secondary antibody into two white paper areas in parallel up and down and adding an NOR and quantum dot marked antibody mixture into a coating layer, folding the paper and permeating the solution into a secondary antibody area; finally, utilizing immunoassay determination to finally quantitatively detect the norfloxacin in the milk through fluorescent signals. The microfluidic paper based sensor disclosed by the invention has the advantages of low cost, simpleness in operation, high flexibility, low detection limit and quickness in detecting NOR.