Alterations　in　protein　(e.g.,　biomarkers)　expression　levels　have　a　significant　correlation　with　tumor　development　and　prognosis;　therefore,　it　is　desired　to　develop　precise　methods　to　differentiate　the　expression　level　of　proteins　in　tumor　cell　lines,　especially　at　the　single-cell　level.　Here,　we　report　a　precise　and　versatile　approach　of　quantifying　the　protein　expression　levels　of　three　tumor　cell　lines　in　situ　using　a　peptide-Au　cluster　probe.　The　probe　(Au(5)Peptide(3))　consists　of　a　peptide　with　a　specific　cell　membrane　epidermal　growth　factor　receptor　(EGFR)　targeting　ability　and　an　Au　cluster　for　both　cell　membrane　EGFR　imaging　using　confocal　microscopy　and　cell　membrane　EGFR　counting　by　laser　ablation　inductively　coupled　plasma　mass　spectrometry.　Utilizing　the　peptide-Au　cluster　probe,　we　successfully　quantify　the　EGFR　expression　levels　of　SMMC-7721,　KB,　and　HeLa　cells　at　a　single-cell　level　and　differentiate　the　EGFR　expression　levels　among　these　cell　lines.　The　peptide-Au　cluster　probe,　with　the　ability　to　differentiate　the　protein　expression　level　of　different　cell　lines,　shows　exceptional　promise　for　providing　reliable　predictive　and　prognostic　information　of　tumors　at　a　single-cell　level.