The development of electronic devices pushes manufacturers to create smaller microchips with higher performance than ever before. Microchip with higher working load produces more heat. This leads to the need of cooling system that able to dissipate high heat flux. Vapor chamber is one of highly effective heat spreading device. Its ability to dissipate high heat flux density in limited space made it potential for electronic cooling application, like Central Processing Unit (CPU) cooling system. The purpose of this paper is to study the application of Al2O3 Nanofluid as working fluid for vapor chamber. Vapor chamber performance was measured in real CPU working condition. Al2O3 Nanofluid with concentration of 0.1%, 0.3%, 0.5%, 1%, 2% and 3% as working fluid of the vapor chamber were tested and compared with its base fluid, water. Al2O3 nanofluid shows better thermal performance than its base fluid due to the interaction of particle enhancing the thermal conductivity. The result showed that the effect of working fluid is significant to the performance of vapor chamber at high heat load, and the application of Al2O3 nanofluid as working fluid would enhance thermal performance of vapor chamber, compared to other conventional working fluid being used before.