Solid-state materials with efficient room-temperature phosphorescence (RTP) emissions have found widespread applications in materials science, while liquid or solution-phase pure organic RTP emission systems has been rarely reported, because of the nonradiative decay and quenchers from the liquid medium. Reported here is the first example of visible-light-excited pure organic RTP in aqueous solution by using a supramolecular host-guest assembly strategy. The unique cucurbit[8]uril-mediated quaternary stacking structure allows tunable photoluminescence and visible-light excitation, enabling the fabrication of multicolor hydrogels and cell imaging. The present assembly-induced emission approach, as a proof of concept, contributes to the construction of novel metal-free RTP systems with tunable photoluminescence in aqueous solution, providing broad opportunities for further applications in biological imaging, detection, optical sensors, and so forth.