Objective: To prepare the double acid etching titanium surface modified with calcium-containing nanoflakes and evaluate its effe cts on osteoblast behaviors in vitro. Methods: Through double acid-etching and hydrothermal treatment with a mixed solution of Ca(OH)2/H2O2, two different calcium-containing nanoflakes(the 1 h and the 6 h treatment groups) were synthesized on the double acid etching titanium surfaces. The sandblasting with large grit and acid etching (SLA) titanium surface was used as the control group, and two double acid etching titanium surfaces modified with different calcium-containing nanoflakes were used as two experiment groups. The micro-topography and surface elemental compositions of different titanium surfaces were observed and analyzed. MC3T3-E1 cells were seeded on samples of the three groups to investigate osteoblast behaviors on different titanium surfaces. Results: Two uniform nanoflake-like structures containing trace calcium were prepared on double acid etching titanium surfaces. Compared with the SLA titanium surface, two modified titanium surfaces significantly up-regulated the adhesion, proliferation, ALP activity and expressions of osteogenic-related genes of MC3T3-E1 cells, especially the 1 h treatment group. Conclusion: The double acid etching titanium surface modified with calcium-containing nanoflakes could promote the adhesion, proliferation and differentiation of osteoblasts.