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In this paper, a novel ammonia detection hybrid film is proposed based on a graphene oxide (GO)/graphene stack, which shows excellent sensing characteristics at room temperature. It is attributed to the cooperation of GO layer serving as molecular capture layer while graphene serving as conductive layer. GO layer is obtained on chemical vapor deposited graphene film by a simple drop-casting method. The prepared GO/graphene hybrid film is directly transferred to the target substrate without any additional transfer vehicle to reduce possible contamination. The success of the transfer depends on the mechanical strength of GO layer. The thickness of GO layer can scale down to 55 nm while sustaining the transfer process. The best ammonia gas sensing performance is obtained at about 275 nm GO layer thickness and the ammonia detection limit is calculated to be 1.5 ppb. In conclusion, the ammonia gas sensing performance of GO/graphene hybrid film can be significantly improved through GO layer thickness optimization.