Nanostructured Ca2CuO3 was synthesized by chemical precipitation method and its application as a catalyst for electrochemical sensing of multi-analytes has been realized for the first time. The composition and phase purity of the synthesized Ca2CuO3 were studied by powder X-ray diffraction, X-ray photoelectron spectroscopy, Scanning and Transmission electron microscopy methods. Ca2CuO3 modified glassy carbon electrode (GCE) exhibited very high catalytic activity towards the oxidation of purine derivatives such as Xanthine (XA), Theophylline (TP), Uric acid (UA), and Caffeine (CF). The Ca2CuO3/GCE could be used over very wide linear ranges of 25 nM - 1.93 mM for UA, 50 nM - 1.62 mM for XA, 250 nM - 2.07 mM for TP and 1.75 μM - 2.11 mM for CF with the corresponding lowest detection limits of 12 nM, 36 nM, 105 nM and 1.72 μM respectively towards the selective determination of the chosen analytes. The developed non-enzymatic sensor was employed to determine all the target analytes in human urine and blood serum samples by standard addition method which displayed high recovery percentages. The developed sensor showed an excellent stability, high selectivity and good reproducibility at physiological pH of 7 making it suitable for pharmaceutical and diagnostic applications.