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Abstract: Phosphopeptide enrichment is essential for the phosphoprotein profiling, due to the low abundance in complex biological samples. Moreover, selective binding of multi‐phosphopeptides over mono‐phosphopeptides is rarely established, but strongly needed in real sample analysis, especially for the investigation of cell behaviors with the multisite phosphorylation cascades. Here two‐dimensional (2D) nanosheets were synthesized of Egyptian blue (CaCuSi4O10), the well‐known ancient pigment, and its analogues (SrCuSi4O10 and BaCuSi4O10), which were employed in the enrichment of phosphopeptides for the first time. Surprisingly, the 2D CaCuSi4O10 nanosheet was highly selective towards multi‐phosphopeptides without enriching the mono‐phosphorylated peptides in a wide range of acidic conditions or buffer compositions. Meanwhile, the SrCuSi4O10 and BaCuSi4O10 nanosheet analogues do not exhibit this unique selectivity. Moreover, the ultrathin and well‐defined 2D morphology, with abundant CaII, of Egyptian blue nanosheet was applied in cortical samples of forebrain specific PDK1 conditional knockout mice and their age‐matched littermate controls, that are associated with Alzheimer's disease. The as‐prepared 2D CaCuSi4O10 nanosheet not only showed specific selectivity, but also exhibited high sensitivity (detection limit of 4×10−7  m). [ABSTRACT FROM AUTHOR]