A novel porous monolithic column was synthesized via in situ free-radical polymerization within a stainless steel column (50 × 4.6 mm i.d.) by using triallyl isocyanurate and methyl methacrylate as co-monomers, ethylene glycol dimethacrylate as a crosslinking agent, 1,4-butanediol, 1- dodecanol, and N, N-dimethylformamide as tri-porogens, and azobisisobutyronitrile as an initiator. The optimized monolithic columns were investigated by scanning electron microscopy, a nitrogen absorption-desorption apparatus, and mercury intrusion porosimetry. The effect of mobile phase composition and temperature on chromatographic behavior was studied in detail as well. The results showed that homemade monolithic columns not only have relative uniform porosity structure, low back pressure, and high thermal stability, but also show high column efficiency, the theoretical plates number of the new polymer monolithic column exceeded 31,000 plates m-1. Good resolution was achieved for HPLC separation of small molecules, including polycyclic aromatic hydrocarbons, isomers of phenols, and the Chinese herbal medicine notoginseng. Methodological validation of six drugs in notoginseng show that correlation coefficients of the linear regressions were in the range of 0.9989-0.9997; the value of relative standard deviation for precision were in the range of 1.37-4.21% for intra-day and 0.34-5.31% for inter-day, respectively; the values of accuracy expressed by recovery were in the range of 100.57-102.51%, 95.18-102.68%, 101.61-102.82%, 100.94-102.40%, 100.99-103.15% and 99.49-103.91% for the six drugs in order, respectively. Taking aromatic hydrocarbons as samples, the relative standard deviation values of the run-to-run (n = 5) and column-to-column (n = 5) based on peak area were < 2.01% and 2.39%, and two RSDs based on retention time were < 2.56% and 2.23%, respectively. The result showed that the polymer monolithic column has good separation ability and high permeability for small molecular substances.