Prostate cancer (PCa), the second most common cancer in men, demands effective early detection strategies. Elevated spermine levels in the prostate tissue contrast with decreased urinary concentrations in PCa patients. Here, we present a novel sensing platform combining differential pulse voltammetry and an extended gate field-effect transistor (EGFET) with a molecularly imprinted polymer|molecular imprinting (MIP) nanofilm for selective and sensitive spermine detection. Key advancements include successfully constructing and characterizing a pseudoreference electrode and a precisely engineered analyte binding interface. The Ag/AgCl pseudoreference electrode exhibited high reliability and reproducibility, optimized to enhance conductivity and minimize interference noises. Electrochemical analysis confirmed successful MIP modification, creating a precise 3D-imprinted binding interface. The platform accurately quantified spermine in artificial urine across concentrations from 0.1 to 1000 ng/mL, achieving a detection limit of 1.23 ng/mL. High selectivity was demonstrated against competing polyamines such as spermidine and histamine. Analysis of electrical properties indicated that spermine binding induced changes in surface potential, altering the metal-oxide-semiconductor field-effect transistor threshold voltage and validating the system’s sensitivity. The system’s superior performance was confirmed with a high imprinting factor (IF ≈ 4.1) and sensitivity 10 times higher compared to nonimprinted polymers. Hill–Langmuir analysis confirmed a strong binding affinity to spermine. Clinical validation using human urine samples from PCa diagnostic evaluations demonstrated high consistency with liquid chromatography mass spectrometry, exhibiting an excellent linear correlation (R2 = 0.97) without statistically significant differences (p-value <0.0001). This study introduces a robust, miniaturized, and cost-effective EGFET-based sensor for spermine detection, offering substantial potential for clinical diagnostics and PCa biomarker monitoring.