A red fluorescent probe, PFB, is designed and synthesized, which presents several remarkable features including a large Stokes shift (85 nm), long emission wavelength, high photostability and good biocompatibility. PFB also displays the solvent-dependent effect, which is verified by the density functional theory (DFT) calculations. Additionally, PFB can act as a fluorescent probe for cellular imaging with high uptake efficiency and highly sensitive detection toward RNA under low optimum concentration (1 μM), which enables it to achieve cancer cell tracing in vitro and tumor growth monitoring in vivo. The results of real-time tracking experiment reveal that PFB can be traced in stained HepG-2 cells for 11 generations and monitor tumor growth for 18 days. This work highlights the potential application of PFB as a promising alternative to the commercial RNA fluorescent probes.