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We report the simultaneous determination of purine molecules with biological significance on pencil graphite electrode (PGE) modified with a composite solution including NiO nanoparticles, multi-walled carbon nanotube (MWCNT), and natural nano-Na-montmorillonite clay (NNaM) using DPV technique. The novel sensor, NiO/MWCNT/NNaM/PGE, achieved simultane-ous determination of xanthine, theophylline, and theobromine at the detection limits 0.077 μM, 0.361 μM, and 0.458 μM with the linear working ranges 0.5-150 μM, 5-200 μM, and 5-250 μM in Britton-Robinson buffer at pH 2.0, respectively. The sensor revealed excellent performance for the simultaneous determination of XT, TP, and TB in three real-world samples.