For an assay to be most effective in point-of-care clinical analysis, it needs to be economical, simple, generalizable, and free from tedious workflows. While electrochemistry-based DNA sensors reduce instrumental costs and eliminate complicated procedures, there remains a need to address probe costs and generalizability, as numerous probes with multiple conjugations are needed to quantify a wide range of biomarkers. In this work, we have opened a route to circumvent complicated multiconjugation schemes using enzyme-catalyzed probe construction directly on the surface of the electrode. With this, we have created a versatile DNA nanostructure probe and validated its effectiveness by quantification of proteins (streptavidin, anti-digoxigenin, anti-tacrolimus) and small molecules (biotin, digoxigenin, tacrolimus) using the same platform. Tacrolimus, a widely prescribed immunosuppressant drug for organ transplant patients, was directly quantified with electrochemistry for the first time, with the assay range matching the therapeutic index range. Finally, the stability and sensitivity of the probe was confirmed in a background of minimally diluted human serum.