Purine bases such as adenine and guanine are fundamental components of nucleic acids and are involved in critical metabolic processes. Abnormal elevations in their concentrations have been associated with various pathological conditions, including cancer. In this study, a highly sensitive voltammetric sensor was developed by modifying a glassy carbon electrode (GCE) with nickel ferrite nanoparticles (NiFe₂O₄ NPs) anchored onto halloysite nanotubes (HNTs), forming a NiFe₂O₄ NPs@HNTs composite. The synergistic integration of redox-active NiFe₂O₄ NPs and high-surface-area, biocompatible HNTs significantly enhanced the electrochemical response by increasing the active surface area, improving electron transfer kinetics, and facilitating strong analyte adsorption. This composite enabled well-defined, irreversible oxidation peaks for both adenine and guanine, with a wide linear detection range from 0.05 to 200 μM. The calculated limits of detection (LODs) were 2.2 nM for adenine and 1.4 nM for guanine, indicating excellent sensitivity. When tested in pre-treated human saliva and serum samples, the sensor achieved recovery rates ranging from 95.7% to 105.2%, with relative standard deviations below 4.21%, confirming its accuracy and reliability in complex biological matrices. These findings highlight the sensor’s strong potential for future clinical applications in purine base monitoring and biomarker-based disease diagnostics.