Engineering nanomaterials for non-invasive electrochemical detection of dopamine (DA) in biological samples has been daunting. We report a novel gold-curcumin (Au-CM) nanocomposite as an electrochemical sensor for real-time ultra-selective detection of DA in urine samples. Gold nanoparticles (∼2–3 nm) encased in porous curcumin (CM) network on the surface of a glassy carbon electrode were synthesized via a galvanostatic method and used as the electrochemical sensor. The modified electrode exhibited excellent sensitivity and selectivity toward DA sensing with a record-low limit of detection (LOD) of 3 pM (signal-to-noise ratio of 3). Our DFT-D3 calculations revealed a higher (by 23.3 kJ mol−1) adsorption energy of DA on the Au-CM nanocomposite than on the bare Au nanocluster. Furthermore, a wide range of detection 1 pM − 400 μM (R
2 = 0.99) was achieved at pH 6. Real-time DA detection was successfully performed in pharmaceutical formulations and urine samples with a single step of dilution with results comparable to clinical values, thus overcoming the complexity of biofluids.