Outer membrane vesicles (OMVs), non-replicating spherical liposomes derived from Gram-negative bacteria, are a promising vaccine platform and multifunctional delivery systems. Their ability to be modified via genetic engineering for the incorporation and display of heterologous proteins enhances their functionality. In this study, we demonstrated a bio-ligation approach to display single-chain variable fragments (scFv) on the OMV surface using the SpyTag/SpyCatcher system. SpyTag-fused scFv, expressed by mammalian cells, bound to OMVs with SpyCatcher-fused Lpp'OmpA after a simple incubation. Biophysical analysis indicated that the conjugated OMVs maintained their physicochemical properties. We used an scFv targeting mucin 1 protein (MUC1) for specific cell targeting. Confocal microscopy revealed that conjugated OMVs specifically bound to and were internalized by MUC1-presenting cells, but not by MUC1-deficient cells. In conclusion, this rapid and efficient bio-ligation system facilitates the display of functional scFv on OMV surfaces, offering a promising approach for targeted delivery to MUC1-expressing cancer cells.