Atomic force microscopy (AFM) was employed to study the formation of Pluronic Polyol F127 oxyethylene99−oxypropylene69−oxyethylene99 (E99P69E99) triblock copolymer micelles on surface-treated silicon substrates. The micellar size determined by dynamic light scattering and small-angle X-ray scattering techniques was 20−30 nm. The two-dimensional micelle size measured by ambient AFM on the modified silicon surfaces was somewhat distorted (20−50 nm in the xy dimension and ∼7 nm in the z dimension) under the environmental conditions present during scanning (i.e., copolymer chain collapsing in air and AFM tip distortion during scanning). Surface treatment was more important than solution concentration for micelle formation on the silicon surfaces. The results could be correlated with the effect of inner capillary surface coating on the resolution of double-stranded DNA capillary electrophoresis by using E99P69E99/1X TBE buffer as the separation medium.