Tumor-seeking fluorescent dyes enable precise lesion localization by recognizing overexpressed receptors, providing critical adjunctive technology for cancer histopathology. However, tumor heterogeneity and poorly understood targeting mechanisms limit their efficacy. To address this, we engineered CETIF6a, a click chemistry-compatible heptamethine cyanine dye, for multi-cancer targeting, intraoperative histopathology, and proteome-wide target identification. Using integrated proteomics, three-dimensional histological electrophoresis, structural biology, and functional validation across multi-cancer types, we demonstrated superior margin delineation (> 90% concordance with H&E) of CETIF6a in cervical squamous, breast, thyroid, and cholangio cancer via molecular weight-resolved fluorescence, eliminating non-covalent background. Identification of the dye’s targets via quantitative proteomics revealing 5–15 times more tumor-specific proteins than paracancerous tissues, with PCNA and DNAJC9 critically involved in cervical squamous carcinoma demarcation covalently bound via nucleophilic substitution at cysteine thiols within hydrophobic cavities. Pan-cancer target synergy through 491 conserved tumor-enriched proteins enriched in ribosomal, proteasomal and metabolic pathways overcoming heterogeneity. Structural convergence of targets (Foldseek clustering, 30% similarity) revealing a dominant functional cluster (63 proteins) enriched in cancer invasion pathways, with ERK1-Cys178 as a key hub stabilized by hydrophobic interactions. CETIF6a’s modifiable scaffold enables dual applications in intraoperative tumor diagnosis and targeted therapy, paving the way for system-wide probe optimization.