In order to non-invasively investigate nucleoplasmic viscosity in real time with good temporal resolution,the present study firstly introduced a new method based on fluorescence correlation spectroscopy (FCS).FCS is a kind of single-molecule technique with high temporal and spatial resolution to analyze the dynamics of fluorescent molecules in nanomolar concentration.Through a time correlation analysis of spontaneous intensity fluctuations,this technique in conjunction with EGFP as a probe is capable of determining nucleoplasmic viscosity in terms of Stokes-Einstein equation as well as its corresponding analysis of the diffusion coefficient for EGFP in the nucleus.The results showed that nucleoplasmic viscosity of ASTC-a-1 cells and HeLa cells were respectively (2.55±0.61) cP and (2.04±0.49) cP at pH 7.4 and 37 ℃,consistent with the results by traditional methods,and nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity.Meanwhile,the real-time analysis of nucleoplasmic viscosity in living cells exposed to hypotonic media proved that FCS could be used to track the changing rheological characteristics of the nucleoplasm in living cells.Taken together,this study suggests that FCS provides an accurate and non-invasive method to investigate the microenvironment in living cells on the femtoliter scale and it can be used as a powerful tool in researches on the dynamical processes of intracellular molecules.