We present a one-step, solid-state synthesis of Al2NiCoO5 nanoflakes (ANC) modified glassy carbon electrode (GCE) for ultra-selective and trace level simultaneous detection of HMIs like copper (Cu2+), mercury (Hg2+), cadmium (Cd2+), and lead (Pb2+) ions in aqua and simulated blood serum samples. ANC/GCE sensor exhibits a low limit of detection (LOD = 3S/m) of 0.00154 ppb, 0.00232 ppb, 0.00261 ppb and 0.00114 ppb towards Pb2+, Hg2+ Cu2+ and Cd2+ ions, correspondingly that are far lower than the perilous limits of HMI concentration reported for blood serum and water samples. The quantitative study and multiple response analysis of the sensor towards each metal ions are accomplished via principal component analysis (PCA) which verifies the selectivity of the ANC/GCE sensor with satisfactory recovery percentages. The superior performance, reproducibility and stability of the sensor are attributed to the electrocatalytic active sites of ANC nanoflakes facilitated by the Ni2+/Ni3+ and Co2+/Co3+ redox couple along with the oxy-functional groups. The sensor was efficacious in detecting the trace level concentrations of HMIs in samples like simulated blood serum, drinking water and tap water with good recovery percentages. This efficiency of the sensor demonstrates it as a promising platform for a broad-spectrum of environmental and bioanalytical applications. © 2022 The Author(s)