In this study, an in-tube solid-phase microextraction column packed with mesoporous TiO2 nanoparticles, coupled with MALDI-TOF-MS, was applied to the selective enrichment and detection of phosphopeptides in complex biological samples. The mesoporous TiO2 nanoparticles with high specific surface areas, prepared by a sol-gel and solvothermal method, were injected into the capillary using a slurry packing method with in situ polymerized monolithic segments as frits. Compared with the traditional solid-phase extraction method, the TiO2-packed column with an effective length of 1 cm exhibited excellent selectivity (alpha-casein/beta-casein/BSA molar ratio of 1:1:100) and sensitivity (10 fmol of a beta-casein enzymatic hydrolysis sample) for the enrichment of phosphopeptides. These performance characteristics make this system suitable for the detection of phosphorylated peptides in practical biosamples, such as nonfat milk.