Tyrosine phosphorylation of proteins plays a vital role in signal transduction pathway. Currently, antibody-based method dominates the tyrosine phosphopeptide enrichment and there is a lack of other general approaches for selective isolation of tyrosine phosphopeptides. However, antibody-based methods are of high cost and biased to certain motifs. Here we developed a chemistry-based method for tyrosine phosphopeptide enrichment. This method utilized the beta-elimination, which only occurs on phospho-serine/threonine residues, to achieve the reverse selection effect. After the dephosphorylation of serine/threonine phosphopeptides a sensitive phosphopeptide isolation method was applied to enrich tyrosine phosphopeptides. In this proof-of-concept study, it showed that the beta-elimination for several standard serine phosphopeptides was completed over 99% while the recovery of tyrosine phosphopeptide remained at around 70% within 20 min. In the further test with 6-protein digests monitored by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, only tyrosine phosphopeptides were detected. Due to the low-cost and unbiased properties, the method has potential applications in tyrosine signaling pathway analysis as an alternative to antibody-based approach.