This study developed and utilized quantum dot conjugates labeled with antibodies (QDλ/Ab) to analyze the heterogeneity of cancer cell populations in co-cultured MCF-7/THP-1 cells, mimicking peripheral blood conditions. We synthesized QDλ with emissions at 450 nm, 525 nm, and 615 nm, confirmed by transmission electron microscopy and X-ray diffraction, suggesting distinct crystal structures and phase transitions. Surface modification with β-mercaptopropionic acid (QDλ/MPAb), polyethylene glycol (QDλ/MPA/PEG) and Streptaviding (QDλ/MPA/PEG/SA) enhanced biocompatibility and enabled specific antibody binding, as evidenced by consistent fluorescence and dynamic light scattering analyses. Flow cytometry and confocal microscopy validated the selective binding of antibodies to cancer markers and revealed significant heterogeneity within the cell populations. This study underscores the potential of QDλ/Ab conjugates in precise cancer biomarker detection and heterogeneity assessment.

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