The development of effective strategies for the massive production of layer-number tunable graphene is of great importance to satisfy the requirements in versatile applications such as energy storage, thermal management, photocatalysis. However, how to prepare the layer-tunable graphene by a simple and efficient way is still a great challenge. Herein, an attempt has been made to exfoliate graphite into layer-tunable graphene by simply soaking the graphite in a binary-component solution composed of H2SO4 and (NH4)2S2O8. In this one-step method, we demonstrate that the layer-number for the as-prepared graphene can be significantly reduced by increasing the exfoliating temperature. An average thickness of ∼20, ∼10, and ∼3 atomic layers can be obtained for the graphene samples exfoliated at the temperature of 30 °C, 60 °C, and 90 °C, respectively. Meanwhile, higher exfoliating temperature not only facilitates the higher efficiency in the exfoliation of graphite, but also achieves a superior conductivity for the prepared graphene. We have demonstrated for the first time that controlling in a sole factor of temperature can effectively tune the layer-number of graphene by a one-step chemical exfoliation method, which will find its great potential in the practical application where the designated property of graphene is required.