This research presents a novel, cost-effective, and scalable approach for the direct detection of myoglobin (Myo) in point-of-care (PoC) applications. In this strategy, redox-active Prussian Blue nanocubes (PBNCs) are applied to a disposable platinum screen-printed electrode (Pt-SPE). Subsequently, a biomimetic sensing layer is generated by electropolymerization of ortho-phenylenediamine (o-PD) in the presence of Myo, which forms molecularly imprinted polymer (MIP) sites by cyclic voltammetry (CV). The electropolymerization process takes place in a potential range of -0.2 V to +0.8 V, for five cycles at a scan rate of 50 mV/s, in a 10 mmol/L o-PD solution. After polymerization, the electrode is incubated in trypsin for 2 h to create Myo-specifically imprinted cavities. The structural and morphological properties of the biomimetic layer were analyzed by Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The direct detection of Myo was analyzed by differential pulse voltammetry (DPV). The results showed a linear response to Myo concentrations ranging from 1.0 ag/mL to 10 ng/mL, a limit of detection (LOD) of 0.76 ag/mL, and a R