Ring-opening reaction of rhodamine spirolactam has been widely applied to construct fluorescent probes. The fluorescence properties of the probe were finely tuned for specific purpose through changing the rhodamine fluorophore. However, the influence on response range and kinetic parameters of the probe during the change has been seldom discussed. Herein, we took pH detection as an example and constructed spirolactam based probes (RLH A-C) with Rhodamine 6G, Rhodamine B and Rhodamine 101. The pK_a values and observed rate constant k_obs of RLH A-C were determined and found to negatively correlated with the calculated Gibbs free energy differences ΔG_C-O and ΔG_TS respectively. The potential applications of RLH A-C in imaging acidic microenvironment were also investigated in cells. We expect the comparison of rhodamine fluorophores will facilitate the quantitative optimization of rhodamine spirolactam based fluorescent probes.