Monodisperse poly(styrene-co-divinylbenzene) (PS-DVB) microspheres with binary porous structures were synthesized using a modified seeded polymerization method. The microspheres had small pores on the surface and big pores in the middle. Using the binary porous PS-DVB (BPPSD) microspheres as packing materials for high-performance liquid chromatography, several benzene analogs were effectively separated in a column as short as 75 mm due to the high surface areas of the stationary phase. Because of the pi-pi affinity interaction between BPPSD particles and analytes, the separation performance of the BPPSD column was better than commercialized C-18 column at the same conditions. Compared to column filled with non-porous PS-DVB particles, the back pressure of BPPSD column could be maintained at especially low level even at high flow rates due to the excellent permeability of the fillers. In addition, approximate baseline separation of C-60 and C-70 was also achieved in the 75 mm BPPSD column.