The objective of this study is to evaluate the possibility of gelatin hydrogel nonwoven fabrics (GHNF) of a cell culture scaffold to formulate 3-dimensional (3D) cell construct. The thickness of cell construct is about 1 mm and the cells inside are live and bio-active, irrespective of their internal distribution. The GHNF were prepared by the solution blow method of gelatin, following by dehydrothermal crosslinking. The GHNF showed a mechanical strength strong enough not to allow the shape to deform even in a wet state. The wet GHNF also showed resistance against repeated compression. After human mesenchymal stromal cells (hMSC) were seeded and cultured, the inner distribution in GHNF, the apoptosis, hypoxia inducible factor (HIF)-1α, Ki67, collagen or sulfated glycosaminoglycan (sGAG) secretion of cells were evaluated. The hMSC proliferated inside the GHNF with time while a homogeneous distribution in the number of cells proliferated from the surface to the 1000 μm depth of GHNF was observed. The number of apoptosis and HIF-1α positive cells was significantly low compared with that of polypropylene nonwoven fabrics with the similar fiber diameters and intra-structure. The GHNF were degraded during cell culture, and completely replaced by collagen and sGAG secreted. It is concluded that the GHNF is a promising cell culture scaffold for 3D cell constructs.