Saxagliptin (Saxa) belongs to a new generation of antidiabetic pharmaceutical compounds used in combination with healthy diet and exercise to lower blood glucose levels in patients with type 2 diabetes mellitus (T2DM). In this work, we report for the first time a molecularly imprinted polymer (MIP) based electrochemical sensor for the determination of Saxa. Computational calculations were performed, based on which five MIPs were synthesized using Saxa as a template, itaconic acid as a monomer, crosslinked with ethylene glycol dimethacrylate and Di methyl sulfoxide (DMSO) as a porogen with different ratios. Non‐covalent interaction (NCI) analysis has been also conducted, and the obtained isosurface analysis was used for graphical visualization of NCI that could occur in real space as well as for the discrimination between hydrogen bond interaction, Van Der Waals attraction and spatial repulsion. The optimized polymer was incorporated as a modifier for designing an electrochemical sensor comprising MIP and Multiwalled carbon nanotubes (MwCNT) within carbon paste electrode (CPE). The operational variables including incubation time, pH, scan rate, and accumulation time were optimized. The sensor showed linearity over the concentration range (1 × 10−9–1 × 10−15 M) with low limit of detection (LOD) 8 × 10−16 and 2 × 10−16 M on using DPV and EIS, respectively. The sensor was successfully applied for pharmaceutical formulations, urine, and human serum samples with recovery range between 97.45–100.64 %.