A one-step and template-free method to prepare uniform SnO2 nanospheres with a mesoporous structure was developed for the applications in phosphopeptide enrichment. The as-synthesized mesoporous SnO2 nanospheres have a large surface area and highly active surfaces for the effective binding of phosphopeptides. Compared with the non-porous SnO2 and commercial TiO2, mesoporous SnO2 nanospheres represent superior performance in the specific trapping of phosphopeptides from both standard protein and complex nonfat milk digests for mass spectrometry-based phosphoproteomic analysis. The feasible synthetic approach and the excellent enrichment performance make the mesoporous SnO2 nanospheres promising in further phosphoproteomic research.