The movement of individual molecules inside living cells has recently been resolved by single particles tracking (SPT) method which is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. Effective treatment of metastatic cancers requires the toxic chemotherapy, however this therapy leads to the multidrug resistance phenomenon of the cancer cells, in which the cancer cells resist simultaneously to different drugs with different targets and chemical structures. P-glycoprotein molecules which are responsible for multidrug resistance of many cancer cells have been studied by cancer biologists during past haft of century. Recently, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. The development of the total internal reflection fluorescent microscope (TIRFM) provided means to monitor dynamic molecular localization in living cells. In this paper, P-glycoproteins (PGP) were labeled with green fluorescent protein (GFP) in living cell membrane of Madin-Darby canine kidney (MDCK) and the TIRFM method was used to characterize the dynamics of individual protein molecules on the surface of living cells. An evanescent field was produced by a totally internally reflected and a laser beam was illuminated the glass-water interface. GFP-PGP proteins that entered the evanescent field appeared as individual spots of light which were slighter than background fluorescence. We obtained high-resolution images and diffusion maps of membrane proteins on cell surface and showed the local diffusion properties of specific proteins on single cells. We also determined the diffusion coefficient, the mean square displacement and the average velocity of the tracked particles, as well as the heterogeneity of the cell environment. This study enabled us to understand single-molecule features in living cell and measure the diffusion kinetics of membrane-bound molecules. Display omitted •The dynamic of P-glycoprotein (Pgp) in living cell membrane of Madin-Darby canine kidney (MDCK) was studied by SMT method.•Pgp molecules labeled with the green fluorescent protein were tracked using the TIRFM.•The diffusion coefficient as well as the heterogeneity of the cell environment were determined.•The mobility of individual molecules was analyzed based on the probability distribution of displacement and trajectories of Pgp molecules.•Intracellular Pgp molecules existed in two types, single and aggregate.