Owing to three major aspects such as crops accelerating potential, evidence providing material during forensic investigation in criminal cases and creator of halogen-assisted carcinogenic by-product trihalomethanes, the monitoring of humic acid is of key attention for scientists working in concerned departments and awareness of general public. Here in this project we used a facile hydrothermal precipitation protocol for synthesising wool-ball like caffeine-assisted copper oxide nanostructures (Caf-CuONSs) using copper nitrate as precursor and caffeine as shape directing material under alkaline influence of ammonia. These nanostructures were characterised through scanning electron microscopy (SEM), Energy-dispersive X-ray (EDX) analysis, Transmission electron microscopy (TEM), X-ray diffraction (XRD), Brunauer–Emmett–Teller (BET) method and Fourier Transform Infra-red (FTIR) spectroscopy for investigation of their various properties. These nanostructures were deposited on the conductive surface of glassy carbon electrode (GCE) followed by nafion coating and employed as highly sensitive and extremely selective sensor for humic acid detection at trace level with three linear ranges, 100–800 ppb, 10–90 ppb and 1–9 ppb with limit of detection (LOD) as low as 68 ppt. As-constructed sensor was applied for amperometric detection of humic acid in tap water with recovery from 98% to 103%, drinking water with recovery from 97.6% to 101.6% and soil sample with recovery from 96.8–102%, collected from Konya, Turkey and Multan, Pakistan, respectively. These recovery ranges were closely matched with the recovery range of 99.2–103.2% exhibited by certified reference material (CRM).