Tooth presents an intriguing option as a bone graft due to its compositional similarity to bone. However, the deproteinized human demineralized tooth matrix (dpDTM), developed to overcome the limited availability of autologous tooth grafts, has suboptimal pore size and surface roughness. This study aimed to fabricate a surface-modified dpDTM using acid etching and collagen coating, followed by in vitro evaluation of physicochemical and biological properties. The dpDTM was modified into two protocols: Acid-modified dpDTM (A-dpDTM) and collagen-modified dpDTM (C-dpDTM). Results demonstrated that A-dpDTM and C-dpDTM had increased pore sizes and rougher surfaces compared to dpDTM. Collagen immobilization was evidenced by nitrogen presence exclusively in C-dpDTM. All groups had a Ca/P molar ratio of 1.67 and hydroxyapatite as the sole constituent, with 65-67% crystallinity. Degradation rates significantly increased to 30% and 20% for C-dpDTM and A-dpDTM, respectively, compared to 10% for dpDTM after 120 days. Cumulative collagen release of C-dpDTM on Day 30 was 45.16 µg/ml. Osteoblasts attachment and proliferation were enhanced on all scaffolds, especially C-dpDTM, which displayed the highest proliferation and differentiation rates. In conclusion, surface modified of dpDTM, including A-dpDTM and C-dpDTM, significantly enhances bioactivity by altering surface properties and promoting osteoblast activity, thereby demonstrating promise for bone regeneration applications.