A new fluorescein-based fluorescent probe for the detection of Au3+ has been developed. This probe was derived from 4′,5′-fluoresceindicarboxaldehyde and exhibited highly selective and sensitive response toward gold(III) ions in aqueous solution. There is a good linear relationship between the fluorescence intensity at 523 nm and the concentrations of Au3+ ranging from 0 μM to 60 μM, which makes it effective for the detection of Au3+. The limit of detection (LOD) was evaluated to be 0.07 μM based on S/N = 3. The fluorescence enhancement was attributed to the irreversible CN bond hydrolysis that occurred to this probe in the presence of Au3+, generating the fluorescent 4′,5′-fluoresceindicarboxaldehyde. Furthermore, the imaging experiments indicated that this probe is cell-permeable and can be used to detect Au3+ within living cell.