Immunofluorescence (IF) is a common method to observe protein distribution and localization at the single-cell level through wide-field fluorescence or confocal microscopy. Conventional protocol for IF staining of cells typically requires a large amount of reagents, especially antibodies, and noticeable investment in both labor and time. Microfluidic technologies provide a cost-effective alternative: it can evaluate and optimize experimental conditions, and perform automatic and high-throughput IF staining on-chip. We employed this method to analyze lysosomal storage disorders (LSDs) based on the expression and morphological distribution of LAMP1 and LC3 in starving cells. With pneumatic valves integrated on-chip, the parallel staining process can be completed within a few hours. The total consumption of each antibody solution for the whole experiment is merely 0.3 μL. This device provides a promising tool for automated high-throughput molecular imaging at cell level that can be applied for diagnostic analysis.