Single crystals of organic semiconductors with perfect crystal structure and minimal density of defects can exhibit high mobility and low spin scattering compared with their amorphous or polycrystalline counterparts. Therefore, these materials are promising candidates as the spin transport media to obtain long spin relaxation times and spin diffusion lengths in spintronic devices. However, the investigation of spin injection and transport properties in organic single crystals is hindered by the inability to construct devices such as single-crystalline organic spin valves (OSVs). Herein, thin and large organic single crystals of 6,13-bis(triisopropylsilylethynyl)pentacene (TIPS-pentacene) were grown on a liquid substrate and transferred to a target substrate carrying ferromagnetic electrodes to construct single-crystalline OSVs. The magnetoresistance (MR) responses of the single crystals were investigated to study their spin injection and transport properties. MR value as high as 17% was probed with an intermediate layer thickness of 269 nm. More importantly, spin transport was still observed in a single crystal of a thickness up to 457 nm, which was much larger than that of polycrystalline thin film. Our research provides a general methodology for constructing single-crystalline OSVs and paves the way to probe the intrinsic spin transport properties of organic semiconductors based on single crystals.