Neutrophils need to migrate through tight tissue spaces to eliminate pathogens, but their movement is often hindered by their large and stiff nuclei. Neutrophil migration is impaired in sepsis patients, but it is unclear whether this defect is related to the deformability of their nuclei. Herein, we designed microfluidic devices with micron-scale narrow slits to simulate biological barriers. This setup allowed us to observe and record neutrophil movement and nuclear deformation in real-time. We also developed a method for morphological analysis to quantify nucleus deformation in numerous individual cells. Our studies showed that neutrophils from healthy individuals could adjust their nuclear shape to squeeze through these constrictions, whereas those from sepsis patients demonstrated less flexibility. Neutrophils with rigid nuclei struggled to pass through narrow gaps and were more likely to rupture under pressure. These findings suggest that the migration defects of neutrophils observed in sepsis may be attributed to the inability of neutrophils to deform their nuclei, highlighting the crucial role of microfluidic technologies in offering new insights into migration defects under pathological conditions.