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As a non-invasive body fluid, urine pH is one of the important biomarkers for diseases such as the kidneys. Therefore, rapid and accurate detection of urine pH is of great clinical significance. A novel fluorescent probe (SPPH-Cl) was developed based on Brooker's merocyanine skeleton for pH detection. The pKa of SPPH-Cl was adjusted to 6.55 using a phenolic hydroxyl ortho substitution strategy, therefore, the fluorescence response range of SPPH-Cl to pH covers the urine physiological pH range (4.6-8.0). SPPH-Cl has excellent water solubility, stable recoverability, wide anti-interference capability, and sensitive reactions to pH fluctuations in pure aqueous solutions. SPPH-Cl has succeeded in applying to monitor the pH of volunteer urine samples based on a standard curve established in artificially simulated urine, and the detection results have accuracy comparable to pH meters. Therefore, this work provided a powerful molecule tool for detecting pH in urine samples., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)