In this endeavor, a novel electrochemical biosensor was designed using multiwall carbon nanotubes (MWCNTs)- and nickel nanoparticles (NiNPs)-embedded anandamide (AEA) imprinted polymer. The NiNPs so synthesized were mortared with MWCNTs and molecularly imprinted polymer (MIP), which enhanced sensitivity and selectivity of the developed sensor, respectively. The characterization methods of AEA-based MIP included X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and Brunauer–Emmett–Teller (BET) analysis, which supported the successful synthesis of the polymer. Electrochemical studies of fabricated sensor were performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy in potentiostatic mode (PEIS). In this first phase of AEA-specific sensor development, MWCNT/NiNP/MIP@SPE was found to successfully discriminate between different concentrations of AEA. The developed sensing platform demonstrated a 100 pM–1 nM linear range with a 0.01 nM detection limit (LOD), 0.0149 mA/pM sensitivity, and 50% stability within 4 months. The sensor demonstrated selectivity toward AEA: although acetylcholine (ACh) and dopamine acted as strong interfering components because of their chemical similarity, the spiked AEA samples demonstrated ∼90% recoveries. Hence, our results have passed the first step in AEA detection at home, although with a clinical setup, future advancement is still required.