Phosphorylation plays vital roles in complex biological processes such as cellular growth, division and signaling transduction. However, due to the low ionization efficiency of phosphorylated peptides, it is still a huge challenge to obtain region-specific phosphorylated peptide distribution by imaging mass spectrometry. To achieve the on-tissue analysis of phosphorylated peptides, we took advantage of a graphene oxide-immobilized enzyme reactor to conduct the in situ digestion, followed by dephosphorylation treatment that removed the phosphate groups and thereby helped to improve the signal intensity of phosphorylated peptides. A visual representation of the phosphoproteome of a human lens was successfully mapped. Results showed that phosphorylated peptides localized mainly in the nucleus region of a healthy lens while the outer cortex is the dominant region for phosphorylated peptides of a cataractous lens.