We investigate the uncertainty associated with the Fluorescence Recovery After Photobleaching, FRAP, which is widely used in the determination of diffusion coefficient for bio molecules. The uncertainty of our FRAP technique stems from the measurement of the spot size and the half time. The uncertainties of the FRAP is evaluated by considering the uncertainty propagation through the measurements of both spot size and the half time. Finally, we suggest an approach to estimate the effective diffusion coefficient by considering slip conditions between the fluorescent beads and the fluid. The diffusion coefficients measured by the FRAP is close to those obtained from the Stokes-Einstein relation together with the slip correction factor rather than that obtained solely by the Stokes-Einstein equation.