Norovirus is a widespread public health threat and has a very low infectious dose. This protocol presents the extremely sensitive mobile detection of norovirus from water samples using a custom-built smartphone-based fluorescence microscope and a paper microfluidic chip. Antibody-conjugated fluorescent particles are immunoagglutinated and spread over the paper microfluidic chip by capillary action for individual counting using a smartphone-based fluorescence microscope. Smartphone images are analyzed using intensity- and size-based thresholding for the elimination of background noise and autofluorescence as well as for the isolation of immunoagglutinated particles. The resulting pixel counts of particles are correlated with the norovirus concentration of the tested sample. This protocol provides detailed guidelines for the construction and optimization of the smartphone- and paper-based assay. In addition, a 3D-printed enclosure is presented to incorporate all components in a dark environment. On-chip concentration and the assay of higher concentrations are presented to further broaden the assay range. This method is the first to be presented as a highly sensitive mobile platform for norovirus detection using low-cost materials. With all materials and reagents prepared, a single standard assay takes under 20 min. Although the method described is used for detection of norovirus, the same protocol could be adapted for detection of other pathogens by using different antibodies.