Hollow silica microspheres (HSMs) with different structures have been synthesized using sacrificed hard template route combined with multiple sol–gel silica shell coating steps. The synthesized HSMs were characterized by SEM and N2 sorption measurements and employed as the stationary phase in thin layer chromatography. Thin layers of HSMs were coated on glass slides and used to separate a model mixture of methyl red and dimethyl yellow. The conditions to achieve the best chromatographic separation efficiency of the synthesized HSMs were optimized. The results show that the organic mixture could be well-separated using the mixture of cyclohexane/toluene/ethanol as the mobile phase. Distilled water was the best mixing agent for the preparation of the thin layer plates. Activation of the plates at 105 °C for 1 h improved the separation efficiency. Under the optimized conditions, the effect of the microstructures of HSMs on the separation efficiency was analyzed. It is shown that the separation efficiency mainly depends on the thickness of the silica shells and the pore size gradient of the nanopores inside the shells. A comparison test shows that HSMs as a stationary phase are advantageous over commercial silica gel in an easy preparation of homogeneous TLC thin plates.