Despite attempts to aid hard tissue regeneration using materials such as gelatin methacryloyl (GelMa), success has been limited. In this study, considering osteogenic differentiation and mineralization of MC3T3-E1 cells, nerve growth factor (NGF) was utilized for osteogenic differentiation by incorporating NGF into GelMA beads. GelMa beads with NGF (GBNGF) or without NGF (GB) were prepared. MC3T3-E1 cells were then encapsulated in GelMA beads fabricated by UV cross-linking. Live/Dead staining results showed that MC3T3-E1 cells in the GBNGF group exhibited more active cell-cell network formation and clearer spindle-shaped cell morphology than those in the GB group. Alizarin Red staining results revealed that MC3T3-E1 cells cultured in the GBNGF group had a higher amount of calcification indicated by a stronger red staining than those cultured in the GB group. Both results demonstrates that NGF could promote cell-cell and cell-matrix interactions, enhancing cell growth and osteogenic differentiation. In conclusion, the incorporation of NGF into GelMA beads can effectively promote growth and osteogenic differentiation of MC3T3-E1 cells. Therefore, GelMA beads containing NGF are expected to be utilized as a new therapeutic strategy for the regeneration of bone defects.