Plasma treatments are becoming a popular method for modifying the characteristics of a range of substrate surfaces. Atmospheric pressure plasma (APP) is cost-efficient, safe and simple compared to high-pressure plasma. This study examined the effects of a low-temperature APP treatment of a hydroxyapatite (HA)-coated Ti (HA-Ti) surface. The APP treatment made the HA-Ti surfaces more hydrophilic without changing surface morphologies. The APP-treated HA-Ti (APP-HA-Ti) surface showed enhanced cell spreading, cell proliferation, and alkaline phosphatase (ALP) levels with more developed cellular networks, and the formed extracellular matrix (ECM) was fused perfectly with the HA substrate than that on the HA-Ti surface. In conclusion, an APP treatment is a potential surface-modifying method that can enhance the cell affinity at the early stages in vitro.