Alginate hydrogels in microtubular structures have great potential to advance three-dimensional (3D) culture, organoid formation, tissue engineering, and cell therapy. To address the need of fabricating consistent, stable hydrogel microtubes for efficient large organoid generation in a simple and quick manner, we have designed needle-in-needle devices to fabricate alginate hydrogel microtubes without any dead volume of the cell-alginate mixture and demonstrated the feasibility of injecting and culturing embryoid bodies in these pre-made hydrogel microtubes. We further used a reverse engineering approach to find out the optimal flow rates and alginate concentration for fabricating pre-made hydrogel microtubes with desired diameter using particular sets of needle-in-needle devices. We established the relationship of the alginate flow rate with diameter and wall thickness of the microtube using mathematic modeling. It offers a way to determine the flow rate for making microtubes with the desired dimension. Additionally, we evaluated the effect of CaCl2 concentration on the diameter as well as stem cell viability. At last, we demonstrated the capacity of fabricating hydrogel microtubes of varying diameters using three sets of needle-in-needle devices and evaluated stem cell growth in these hydrogel microtubes. It provides a new avenue to accessible, repeatable, scalable, and easy to use pre-made ‘off-the-shelf’ hydrogel microtubes for 3D cell culture including, but not limiting to stem cells.