Abstract: Imprinted polymers were prepared using bulky and layer-coated silica nanoparticles to analyze trace glycine in human urine. In the layer-coated silica nanoparticle-imprinted polymer, the polymerizable double bonds were first grafted on the surface of silica nanoparticles throgh silylation to induce the selective occurrence of surface polymerization. Then, glycine templates were imprinted into the polymer-coated layer through interaction with functional monomers. The molecularly imprinted polymer (MIP) and SiO2-MIP were tested in batch experiments to evaluate their binding properties and then used as SPE sorbents for the selective removal and pre-concentration of glycine. The glycine-imprinted polymer nanoparticles presented higher selectivity and affinity to glycine than bulky imprinted polymer. Glycine was directly extracted from spiked human urine. MIP and SiO2-MIP allowed glycine to be pre-concentrated while removing interfering compounds from the matrix. SiO2-MIP showed high efficiency for the enrichment of glycine in real samples