A method for the fast and accurate measurement of diffusion coefficient of minute proteins was developed. A protein sample plug was introduced into the capillary and forced to flow through a capillary coated dynamically by poly(diallyldimethylammonium chloride). Its concentration profile was then monitored by the UV absorption detector mounted on Beckman MDQ capillary electrophoresis system. Accurate diffusion coefficient was obtained. The key conditions for the accurate measurement include: (1) systematic peak should be developed in a laminar flow; (2) radius diffusion is negligible compared with the longitudinal diffusion; (3) the capillary is well thermostated and (4) the solute should not be overloaded. In common case, the deviation of the measurement was kept within 4%.