Phosphorescence analyses have attracted broad attention due to their remarkable merits of the elimination of auto-fluorescence and scattering light. However, it remains a great challenge to develop novel materials with uniform size and morphology, stability, long lifetime, and aqueous-phase room temperature phosphorescence (RTP) characteristics. Herein, monodisperse and uniform RTP nanobeads were fabricated by an in situ covalent hybridization of carbon dots (CDs) and dendritic mesoporous silicon nanoparticles (DMSNs) via silane hydrolysis. The formation of Si-O-C and Si-C/N covalent bonds is beneficial for the fixation of vibrations and rotations of the luminescent centers. Specially, the nanopores of DMSNs provide a confined area that can isolate the triplet state of CDs from water and oxygen and thus ensure the occurrence of aqueous-phase RTP with an ultra-long lifetime of 1.195 s (seen by the naked eye up to 9 seconds). Through surface modifying folic acid (FA), CDs@DMSNs can serve as a probe to distinguish different cell lines that feature varying FA receptor expression levels. In addition, taking MCF-7 as the model, highly sensitive and quantitative detection (linear range: 103-106 cells per mL) has been achieved via an RTP probe. Furthermore, their potential applications in cellular and in vivo time-gated phosphorescence imaging have been proposed and demonstrated, respectively. This work would provide a new route to design CD-based RTP composites and promote their further applications in the medical and biological fields.