Fe3+ plays a crucial role in many vital cell functions and its detection has attracted considerable attention. In this work, a water-soluble rhodamine-based chemosensor RL has been designed and synthesized as an “off-on” chemosensor for Fe3+ detection. Upon the addition of Fe3+, RL displayed obvious color change, quick fluorescence enhancement, high sensitivity and anti-interference capacity over other metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+). The detection limit for Fe3+ was calculated to be 0.28 μM with the binding constant Ka to be 4.67 × 108 M−2. Furthermore, an application of RL in the imaging of HeLa cells exposed to Fe3+ was also successfully demonstrated.