Ozone is currently being considered as a potential oral antiseptic agent because it is highly antimicrobial and does not induce microbial resistance. In this study, we demonstrated that an optimal dosage of ozone gel enhanced the proliferation, type 1 collagen production, and alkaline phosphatase (ALP) secretion of Saos-2 cells in vitro. Proliferation of Saos-2 cells was assessed by MTT and DNA synthesis assays. Type 1 collagen production and ALP secretion were evaluated using enzyme-linked immunosorbent assay (ELISA) and ALP assays. The cells were treated with/without 0.05, 0.5, 5 ppm ozone gel for 24 h. Ozone gel (0.5 ppm) significantly induced the proliferation of Saos-2 cells. At this concentration, ozone gel enhanced type 1 collagen production and ALP secretion. The results indicated that ozone gel controls the cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers.