The objective of this project was to develop a DNA hybridization sensor that can detect the presence of E. coli and reveal its resistance to the drug gentamicin. This probe will enable rapid and user-friendly diagnostics of E. coli infections and analysis of bacterial gentamicin-susceptibility profile by interrogation of a fragment of E. coli 16S rRNA bearing a substitution in the gentamicin-resistant cells. The sensor is promising for the point-of-care use to provide a timely UTI diagnostic solution. A quick diagnosis of E. coli infection and antibiotic resistance is crucial for treatment. To design a hybridization probe, we proposed a split approach for target interrogation and catalytic activity of a peroxidase-like deoxyribozyme (PDz) as a signal reporter. PDz contains a series of guanine residues in a strand and has been shown to form a parallel guanine-quadruplex (G4). This G4, with the addition of a hemin cofactor, catalyzes the reaction similar to that of horseradish peroxidase. If a colorless organic indicator is added to the G4-PDz-hemin containing solution and mixed H2O2, a colored oxidation product is formed (e.g., a dark blue/green). The color change reports the presence of the catalytically active G4, which occurs only when the nucleotide sequence of the target is a perfect match. When the target is not a perfect match, for example, in the case of the drug-causing nucleotide substitution, the G4 does not form, and there is no color change. The probes tested in this paper show promising results of such a sensor by being able to catalyze the described colorimetric reaction to generate a strong signal in the presence of a "gentamicin-susceptible" target and show selectivity against the "gentamicin-resistant" target.