RF sputtering process was applied to produce thin hydroxyapatite(HAp) films on Ti-6Al-4V alloy substrates. The effects of different heat treatment conditions on the hardness between HAp thin films and Ti-6Al-4V alloy substrates were studied. Before deposition, the Ti-6Al-4V alloy substrates were heat treated for 1h at $850^{circ}C;under;3.0{ imes}10^{-3}torr$, and after deposition, the HAp thin films were heat treated for 1h at $400^{circ}C,;600^{circ}C;and;800^{circ}C$ under the atmosphere, and analyzed FESEM-EDX, FTIR, XRD, nano-indentor, micro-vickers hardness, respectively. Experimental results represented that the surface defects of thin films decreased by relaxation of internal stress and control of substrate structure followed by heat treatment of substrates before the deposition, and the HAp thin films on the heat-treated substrates had higher hardness than none heattreated substrates before the deposition, and the hardness properties of HAp thin films and Ti-6Al-4V alloy substrates appeared independent behavior, and the hardness of HAp thin films decreased by formation of $VTiO_3(OH),;{ heta}-Al_{0.32}V_2O_5,;Al_{0.33}V_2O_5$.