Quantitatively visualizing the thermal dehydration in metal-organic frameworks (MOFs), especially at the single-particle level, is still challenging, hindering a deeper understanding of the reaction dynamics. Using in situ dark-field microscopy (DFM), we image the thermal dehydration process of single water-containing HKUST-1 (H2O-HKUST-1) metal-organic framework (MOF) particles. DFM maps the color intensity of single H2O-HKUST-1, which is linearly correlated with the water content in the HKUST-1 framework, enabling a direct quantification of several reaction kinetic parameters of single HKUST-1 particles. Interestingly, when H2O-HKUST-1 is transformed into deutoxide (D2O)-containing HKUST-1, the corresponding thermal dehydration reaction displays higher temperature parameters and activation energy but shows a lower rate constant and diffusion coefficient, revealing the isotope effect. The significant variation of the diffusion coefficient is also confirmed by molecular dynamics simulations. The present operando results are anticipated to provide valuable guidelines for the design and development of advanced porous materials.