The development of induced pluripotent stem cell (iPSCs) has opened unprecedented opportunities for biomedical applications, but poorly defined animal-derived matrices yield cells with limited therapeutic value. Considerable challenges remain in improving cell-culturing approaches to create the conditions for iPSCs' reliable expansion. Herein we report the development of a chemically defined, artificial three-dimensional (3D) microniche for iPSCs’ growth and reliable expansion, constructed with degradable polyethyleneglycol-co-polycaprolactone and RGDfk-functionalized dendritic polyglycerol precursors according to bioorthogonal strain-promoted azide-alkyne cycloaddition by droplet-based microfluidics. This compatible microniche can allow for the robust production of iPSCs that maintain high pluripotency expression and excellent viability without pathogen or immunogen transfer risks. This microniche technology shows great promise in enabling iPSCs to achieve their full therapeutic potential.