This paper described the optimized design, synthesis and application of a novel rhodamine thiospirolactam derivative as an ‘off-on’ fluorescent probe for the detection of Hg²⁺ in aqueous samples. The ‘off-on’ fluorescence and color signal change of the probe is based on an Hg²⁺-triggered domino reaction which brings on the opened-ring form of the rhodamine spirolactam to regain the conjugated system of the rhodamine skeleton. In the well designed probe, the thiospirolactam serves as both Hg²⁺ binding unit and electron-defect carbon centre, a phenolic hydroxyl with very strong nucleophilicity after deprotonation is chosen as the attacking unit, and a benzene ring is introduced on the linker to afford steric effects, which benefits an efficient nucleophilic reaction, with a high sensitivity towards Hg²⁺. It exhibits a stable response for Hg²⁺ from 1.0 × 10⁻⁸ to 1.0 × 10⁻⁶ M, with a detection limit of 3.0 × 10⁻⁹ M. The response of the probe to Hg²⁺ is highly selective and pH-insensitive, with a fast response time. All these unique features make it particularly favorable for cellular Hg²⁺ imaging applications. It has been preliminarily used for highly sensitive monitoring of Hg²⁺ levels in living cells with satisfying resolution.