Human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) have shown great potential in tissue repair and regeneration. However, their scalable production and functional quality are still limited by current expansion technologies. In this study, we propose a production technology for hMSC-EVs based on three-dimensional (3D) microbead culture, which enhances the secretory behaviour of hMSC. Fixed number of MSCs were encapsulated in Matrigel at appropriate densities and printed into 3D microbeads by the custom automated microfluidic bead-jet printing technique. Compared with 2D culture group, EVs derived from 3D hMSC microbead had smaller size and increased yield by 20-fold, and the actin depolymerisation of the cell may be an important mechanism for enhancing EV secretion. Further analysis confirmed that the EVs derived from 3D hMSC microbead exhibited enhanced angiogenic and proliferative capabilities, which promoted the viability and tube-forming capacity of human umbilical vein endothelial cells (HUVEC). In conclusion, this automated microfluidic microbead encapsulation technology increased the yield and therapeutic effect of hMSC-EVs and provides a platform for scalable EV production of regenerative therapies.