In this investigation, water-soluble N-Acetyl Cysteine Capped-Cadmium Telluride QDs (NAC/CdTe nanocrystals), utilizing N-acetyl cysteine as a stabilizer, were prepared to assess their potential in differentiating between DNA extracted from pathogenic bacteria (e.g. Escherichia coli isolated from urine specimen) and intact DNA (extracted from blood of healthy individuals) for biomedical sensing prospective. Following the optical characterization of the synthesized QDs, the XRD analysis illustrated the construction of NAC-CdTe-QDs with a grain size of 7.1 nm. The prepared NAC-CdTe-QDs exhibited higher PL emission features at of 550 nm and UV-Vis absorption peak at 300 nm. Additionally, the energy gap quantified via PL and UV–Vis were 2.2 eV and 2.3 eV, respectively. The interconnection between the synthesized QDs and the different types of the extracted genomic DNA (both Escherichia coli and healthy subjects) was analyzed optically. This is resulted in a clear shift in the maximum fluorescence emission intensities (observed at 533 nm for an Escherichia coli DNA and 541 for healthy DNA). Overall, the present study findings suggest that prepared QDs could be employed as probes for the detection of pathogenic bacteria DNA from that of healthy subjects.