Myo-inositol (MI) is a secondary messenger that can be used to treat a variety of metabolic syndromes (MS), from diabetes to cancer, and ongoing research is reassuring a bright future for therapies. In this study, two molecular imprinting polymer (MIP) sensor matrixes were created and investigated for the determination of MI; (i) 3-aminophenylboronic acid (3-APBA) electropolymerization with MI as the template on the SPCE surface (ii) gold nanoparticles (AuNPs) initially electrodeposited on a screen-printed carbon electrode (SPCE) surface and further the molecularly imprinted poly-3-aminophenylboronic acid (PAPBA) sensor film was electropolymerized with MI on the modified AuNPs/SPCE surface. Here, the surface modification with AuNPs on SPCE better enhances the electrochemical signal compared with unmodified SPCE. The electrochemical characterization of molecularly imprinted sensors was evaluated via Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV) techniques. The morphological changes of the modified electrodes were analyzed by Scanning Electron Microscope (SEM). The performances of the imprinted sensors (such as extraction of the template, rebinding conditions, and interference study) were investigated and optimized. The overall analytical data of the MI/PAPBA/AuNPs/SPCE based imprinted sensor (linear range: 500 nM to 60 μM; limit of detection: 1.0 nM; sensitivity: 0.042 μA μM−1) was better than the MI/PAPBA/SPCE (linear range: 500 nM to 50 μM; limit of detection: 4.7 nM; sensitivity: 0.019 μA μM−1). Further, the MI/PAPBA/AuNPs/SPCE imprinted sensor was successfully validated by applying for the sensitive and selective detection of myo-inositol in human plasma samples, and the results agreed well with the HPLC literature data.