Objective From the perspective of biophysics and immunology, the effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on biophysical characteristics, cytoskeleton and migration ability of mouse derived dendritic cells (DCs) were analyzed, so as to explore the effect of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) on immune function of DCs and its potential mechanism. Methods The bone marrow-derived monocytes from C57BL/6J mice were isolated and induced to differentiate into immature dendritic cells (imDCs) by 20 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4), After 6 days, 100 ng/mL lipopolysaccharide was added to induce mature dendritic cells (mDCs). Further the morphological observation and positive rate of CD11c in imDCs and mDCs were analyzed, Then at different concentrations of EPA and DHA (0-60 μmol/L), the cell viability and apoptosis of DCs were detected by cell counting kit-8 (CCK-8) kit and flow cytometry. After the optimal concentration of EPA and DHA were determined, the changes of the membrane fluidity, electrophoretic mobility (EPM) and osmotic fragility of DCs were separately detected by the fluorescence polarization, cell electrophoresis and concentration gradient. The expression of filamentous actin (F-actin) was detected by the immunofluorescence. Finally, the migration ability of DCs was detected by the Transwell system. Results The positive rate of CD11c in DCs was about 80%. The viability of DCs decreased in a dose-dependent manner under the action of EPA and DHA of different concentrations, which didn’t induce the apoptosis of DCs. Under the action of 50 μmol/L EPA and DHA, the osmotic fragility and EPM of DCs significantly decreased, and the membrane fluidity significantly increased. The expression of F-actin in DCs obviously increased, and the migration rate of cells obviously decreased. Conclusions ω-3 PUFAs may affect the cytoskeleton structure and biophysical characteristics of DCs, inhibit the migration, and then affect its immune function.