Porous silica spheres were prepared via an acid-basic catalytic sol-gel method. The effects of some factors including water amount, ethanol amount, rotary evaporation temperature, stirring speed and porogen species on the morphology and particle size distribution of the silica spheres were investigated. The obtained silica spheres were characterized by electron microscope, BET measurements, titration method and infrared spectra. Moreover, after simple sedimentation and sorting, the silica spheres were functionalized and four packings were obtained, including C18 bonded silica gel, hydrophilic C18 bonded silica gel, aminopropyl bonded silica gel and ethylenediamine-N-propyl bonded silica gel. Results showed that the optimal conditions were as follows: n(H2O): n(TEOS)=6/4, V(EtOH):V(TEOS)=3/5, rotary evaporation temperature of 57 ℃, stirring speed of 2190 r/min and porogen of DMF. The obtained silica spheres were mostly regular-ball, the specific surface area was 464.11 m2/g and pore volume was 1.14 m3/g. The average pore size was 9.81 nm with a narrow pore size distribution and the concentration of Si-OH groups was up to 0.5450 mmol/g. The column efficiency of C18 bonded silica gel and hydrophilic C18 bonded silica gel were up to 53474/m and 86984/m (toluene), respectively, which had good resolution of analytes. The ion exchange capacity of aminopropyl bonded silica gel and ethylenediamine-N-propyl bonded silica gel were 1.44 and 1.22 mmol/g, respectively. Their absorption capacity to toluene-p-sulfonic acid was up to 240.8 and 217.6 mg/g.