An experimental investigation is performed into the two-phase spray cooling heat transfer performance of Al2O3-water nanofluid given three different nanoparticle concentrations (0, 0.001 and 0.05 vol%) and three different spray operating times (10, 20 and 30 hours). The results show that for an Al2O3 concentration of 0 vol% (i.e., pure DI water), the spray operating time has a negligible effect on the heat transfer performance. However, for Al2O3 concentrations of 0.001 and 0.05 vol%, the heat transfer coefficient decreases with an increasing spray operating time. For the nanofluid with 0 vol% Al2O3, the spray operating time has only a small effect on the surface hydrophilicity. However, for the nanofluids with nanoparticle concentrations of 0.001 and 0.05 vol%, respectively, the surface hydrophilicity increases with an increasing spray operating time. Furthermore, the surface hydrophilicity also increases with an increasing nanoparticle concentration for a given spray operating time. The energy dispersive X-ray spectroscopy (EDS) results reveal the presence of a nanoparticle layer on the sprayed surfaces with a higher heat flux. The thickness of the deposited layer increases with an increasing spray operating time, and leads to a lower heat transfer performance and a higher surface hydrophilicity as a result.