A convenient and effective 'ultrasonic dispersion method' was used to fabricate vector/DNA complexes encapsulated microspheres. Polyamidoamine (PAMAM) dendrimer/DNA complexes protected by a water-soluble polymer, poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), were encapsulated in a polymer film mainly composed of cholic acid functionalized star poly(DL-lactide), which degraded through surface erosion mechanism with a fast degradation rate. The PAMAM/DNA complexes encapsulated polymer film was then immersed in ethanol and ultrasonicated to afford the microspheres. The in vitro gene transfections showed PAMAM/DNA complexes protected by PHEA exhibited a much higher transfection activity compared with PAMAM/DNA complexes without the protection by PHEA. The expressions of pGL3-Luc in HEK293 cells could be effectively mediated by the polymer film and microspheres with the presence of PHEA. The ultrasonic dispersion method, which did not involve toxic organic solvents, could keep the bioactivity of DNA and offer good control over the size of microspheres.