We propose an original concept for a sandwich immunoassay that is completely performed on-chip using streptavidin-coated beads as substrate. The latter are electrostatically self-assembled on aminosilane micropatterns at the bottom of a microfluidic channel. We use mouse IgG diluted in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) solution as target antigen. The fluorescent sandwich immunocomplex is formed on the beads during the operation of the chip both in stop-flow and continuous-flow modes. Target mouse IgG antigen is detected down to a concentration of 15 ng/mL in stop-flow mode and 250 pg/mL in continuous-flow mode, using only 1300 nL of sample volume. We also demonstrate the possibility of simultaneous detection of two different antigens in a PBS−BSA solution using a dual microfluidic channel structure.