A molecularly imprinted polymeric monolith (MIPM) was prepared by in situ polymerization using styrene, glycidyl methacrylate and methacrylic acid as monomers, divinylbenzene and triallyl isocyanurate as cross-linking agents, and ceramide III as print molecule. The texture, pore size distribution, mobile phase flow characteristic, and chromatographic performance of the MIPM and a control monolith synthesized without the print molecule were examined, respectively. The results showed that using ceramide III as print molecule significantly affected the pore structure and pore distribution of the monolith, and greatly improved the retention of ceramide III and its analogues used in cosmetics as well. The retention of ceramide III on the MIPM could be reduced by increasing the ratio of chloroform to hexane in eluting buffer. The workability of the MIPM was firstly demonstrated through the separation of a model lipid mixture containing ceramide III and ergosterol, the main sterol impurity in yeast lipid extracts. The application of the ceramide III imprinted monolith to the isolation of ceramides from yeast lipid extracts was attempted and resulted in a considerable enrichment of ceramides, as shown by FTIR analysis. This indicates the potential of ceramide III imprinted monolith synthesized in the present study in the on-line solid-phase extraction of ceramides from yeast.