This study investigated the spray cooling heat transfer performance of Al2O3-water nanofluid given four different subcooling degrees (0 °C, 10 °C, 20 °C, and 30 °C). The results showed that the subcooled nanofluids were ranked in order of a reducing spray cooling heat transfer performance as follows: 20 °C, 10 °C, 0 °C, and 30 °C. On average, the heat transfer coefficient obtained using the nanofluid with 20 °C subcooling was around 8.3%, 8.6%, and 15.6% higher than that obtained with 10 °C, 0 °C, and 30 °C subcooling, respectively. However, the heat transfer performance decreased with an increasing spray operating time. The scanning electron microscopy observations showed that the reduction in the heat transfer coefficient was the result of a gradual increase in the thickness of the nano-adsorption layer on the heated surface as the spray operating time increased.