Zirconia ceramics has a bioinert property with low bioactivity. So, it is necessary to improve its low bioactivity by the surface modification using effective coating methods. In this study, we fabricated the hydroxyapatite-coated zirconia substrate by room temperature spray processing to improve the bioactivity of the zirconia implant and investigated its coating effect on the biological performance of zirconia substrate via an in vitro test in simulated body fluid (SBF) solution. Before the room temperature spray coating was completed, size-controlled hydroxyapatite powder that had an average size of 4.5 μm, was obtained by the calcination and milling of a commercial powder. By controlling the processing parameters, such as spraying distance, and deposition cycles, we fabricated homogeneous and dense hydroxyapatite coatings on zirconia substrate. Surface morphology, coating thickness, and microstructure were dependent on deposition cycles, and were related to surface roughness and bioactivity. Zirconia substrates with wave-patterned and roughened hydroxyapatite coatings demonstrated high bioactivity in their in vitro tests. Via the immersion test in an SBF solution, surface dissolution and new precipitates of hydroxyapatite were observed on coated zirconia substrate, indicating the degree of bioactivity.