In this work, we demonstrate trace level electrochemical detection of atrazine in human blood serum samples based on lead-free double perovskite material Al2NiCoO5 (ANCO) modified glassy carbon electrode (GCE). The SEM micrographs confirm the nanoflakes morphology of the ANCO synthesized via solid-state technique while XRD, Raman and FTIR analysis reveal the crystal structure, vibrational modes and the existence of OH− groups at the surface area of the ANCO nanoflakes. The as-fabricated ANCO/GCE exhibits a low limit of detection (LOD) of 0.3 ppb and a linear range of 0.5 ppb – 100 ppb atrazine with a sensitivity of 0.929 μA/ppb cm2. This enhanced performance of ANOC/GCE is ascribed to the electrocatalytic active sites of ANOC nanoflakes facilitated with Ni2+/Ni3+ and Co2+/Co3+ redox couple along with the oxy functional groups. The sensor exhibits an outstanding selectivity towards atrazine in presence of 100 folds of Na+, Ca2+, bovine serum albumin (BSA) and human serum albumin (HSA). The sensor is successful in the detection of atrazine in human blood serum samples with outstanding recovery percentages from ∼97.05 to ∼103.27 % thus making it an ideal platform for developing a wide range of electroanalytical sensors in biological samples. © 2020 Elsevier B.V.