Dielectrophoretic behaviors and assembly of a binary suspension in aqueous media are examined in the presence of nonuniform alternating current (AC) electric field. A peculiar low-frequency threshold and dielectrophoresis (DEP) crossover frequency determine the applicable frequency window for binary assembly under positive DEP, which can be effectively tuned by medium conductivity and particle size, suggesting that the dynamic double-layer effect is responsible for the inter-facial polarization of micrometer to submicrometer-sized particles in aqueous suspensions. Strong effects of AC-field frequency, medium conductivity, and size ratio on binary assembly morphology have been observed. A frequency-medium conductivity phase diagram is obtained to illustrate the morphological transition of assembled colloidal aggregates from segregated, ordered assemblies to inverted segregation with the appearance of amorphous phases upon increasing frequency and/or medium conductivity, which is a direct consequence of the competition between DEP and hydrodynamic mobility. Significantly, our results demonstrate a rapid method to form hybrid nanostructured materials.