The aim of this study was to compare human dental pulp stem cells’ (DPSCs) attachment, proliferation and osteogenic differentiation on allogenic and synthetic biphasic bone granules. In this in vitro study, two types of bone granules were used: allograft [freeze-dried bone allograft (FDBA)] and biphasic granules [hydroxyapatite/beta-tricalcium phosphate (HA/β-TCP)]. By isolation of DPSCs, their attachment to bone granules was observed by scanning electron microscope (SEM) at day 1 and 7 of cultivation. Vital cells were measured by MTT assay at 1, 3, and 7 days of cell culture. Comparison of vital cells at different time points was considered as cell proliferation. Finally, differentiation of DPSCs was evaluated by measurement of alkaline phosphatase (ALP) activity 3, 7, 14, and 21 days after cell seeding in standard and osteogenic media. Data were analyzed using two-way ANOVA with a significant level of 0.05. Attachment of DPSCs on FDBA granules seemed relatively stronger. The number of cells (based on MTT values) and ALP activity of the cells cultured on both study groups increased between time points (p ≤ 0.001). FDBA granules had more cells compared to HA/β-TCP granules (p < 0.001). There was no significant difference between ALP activity of two study groups cultured in the standard medium (p = 0.347) and they were both higher than the control group (p < 0.05). In the osteogenic medium, FDBA group had significantly higher ALP activity compared to HA/β-TCP (p = 0.035) and control (p = 0.001) groups while there was no significant difference between ALP activity of HA/β-TCP and control groups (p = 0.645). In conclusion, current in vitro study revealed that FDBA granules have more potential in supporting DPSCs attachment and proliferation and inducing their ALP activity compared to HA/β-TCP granules. Therefore, FDBA could serve as a proper bone substitute material.