We investigated a water-soluble conjugated polymer (WSCP) with pendant disulfide linkages to poly(ethylene glycol) (PEG) chains, which is a highly efficient ratiometric probe with solubility-induced fluorescence conversion for thiol detection. This WSCP was doped with a low-bandgap fluorophore, 1,4-dithienyl benzothiadiazole (DBT), and was modified with PEGs by disulfide linkages to increase its water solubility. The free probe exhibited good solubility in aqueous solution (28 mg/mL) and showed purple fluorescence because of the low doping ratio of DBT. The separation of water-soluble PEG chains from the conjugated backbone induced by the cleavage of the disulfide linkages would lead to a significant decrease of the water solubility of the probe. The combined utilization of scanning electron microscopy, dynamic light scattering, and fluorescence spectrophotometer further confirmed that decreased solubility produced an aggregation of the hydrophobic conjugated backbone and subsequently increased fluorescence resonance energy transfer efficiency from the conjugated backbone to DBT which manifested as fluorescence conversion from purple to red. The fluorescence ratiometry (I-628/I-420) of the probe varied from the lowest value of 0.095 to 1.15 (12-fold maximum enhancement). The detection limit was 2.56 mu g/mL (0.021 mM). The WSCP probe was confirmed to be a good sensing material with high selectivity for thiols by examining various biological molecules. We also successfully achieved the imaging of intracellular thiols in HeLa cell. Considering that the disulfide could be replaced by other cleavable linkages, such a fluorescence ratiometry induced by decreased solubility could be utilized for detecting other chain-cleavable biomolecules, which would contribute to the development of new probes based on conjugated polymers.