The minimum mode spot size of a three-layer dielectric optical waveguide was studied. Based on the mode-field solutions expression of Maxwell function, The influences of mode polarization, waveguide size, dielectric coefficient difference, wavelength and asymmetry on mode spot size, according to its specified definition are investigated. The analysis and simulation results indicated that there existed a minimum mode spot size (MMSS) in a three-layer dielectric optical waveguide. As to the symmetric waveguide, the MMSS of TE mode is inversely proportional to the square root of dielectric coefficient difference. However the MMSS of TM mode is dependent on the specified dielectric coefficients of waveguide and larger than that of TE mode. Both of them are proportional to the wavelength of guided wave. As to the asymmetric waveguide, the shape of mode spot is also asymmetric. The MMSS has the same dependences on dielectric coefficient difference as symmetric one. Besides, it decreases at first and then increases with the increasing of waveguide symmetry factor.