Based on the derivatization of R-phycoerythrin (R-PE) with o-phthalaldehyde (OPA), a novel fluorescence ratio probe for the detection of o-phthalaldehyde was constructed. The primary amino groups of R-phycoerythrin were exposed after unfolding by sodium dodecyl sulfate (SDS). In the alkaline environment of β-mercaptoethanol, a blue fluorescent isoindole compound could be derived by reacting with o-phthalaldehyde. In this probe, the fluorescence signal of R-phycoerythrin gradually weakened with the increase of the concentration of o-phthalaldehyde, whereas the blue fluorescence signal was gradually enhanced, and a strong-weak fluorescence change constituted a ratio fluorescence probe. The results show that under the carbonate buffer condition of pH 10.5, the optimal detection time of the probe is from 3 to 6 minutes, the linear detection range is from 5 to 100 μg/mL, the correlation coefficient is 0.992, and the limit of detection is 1.7 μg/mL. The proposed fluorescent probe can sensitively detect the OPA in disinfectant.