DNA-protein bioconjugation is an appealing target-tracking strategy. The new capability of DNA molecule as a biological nanomaterial endows unique fluorescence and physicochemical properties to be applied in bioimaging. Progression in targeted imaging is contingent on the conjugation of diagnostic nanoparticles to biomolecular signatures, particularly antibody-based ligands. Here, we have reported our recent experience, DNA-dot synthesis and characterization, the covalent conjugation of DNA-dot to goat F(ab’)2 IgG and Epidermal Growth Factor Receptor (EGFR) antibodies, DNA-dot@antibody coupling confirmation, and fluorescent targeted imaging of lung cancer cell line. As a result, the average size of DNA-dot was 4.5-5 nm which was conjugated to amine-rich antibodies with returned PO groups on the DNA-dot surface via PN bond. The synthetic DNA-dots were conjugated to the goat F(ab’)2 IgG and tested for fluorescent detection usability by indirect Dot-blot assay. Also, DNA-dot@EGFR conjugates identified lung cancer cells with 84-92% specificity and 100% sensitivity in five concentrations, associated with 0.0025 to 0.04 g 100 μL DNA-dot. The results demonstrated that bioconjugated DNA-dot can do the diagnosis profiling of molecular biomarkers. Generally, DNA-dot bioconjugation with antibody is implemented within two days and biomarker detection takes one day. Consequently, DNA-dot@antibody is potentially a toxic-free, swift, and efficient method of antibody labeling that opens up new horizons in fluorescent nanoimaging in the field of cancer cell detection.Copyright © 2020 Elsevier B.V. All rights reserved.