The detection of glucose is crucial for diagnosing diseases such as diabetes and enables timely medical intervention. In this study, a disposable enzymatic screen-printed electrode electrochemical biosensor enhanced with machine learning (ML) for quantifying glucose in serum is presented. The platinum working surface was modified by chemical adsorption with biographene (BGr) and glucose oxidase, and the enzyme was encapsulated in polydopamine (PDP) by electropolymerisation. Electrochemical characterisation and morphological analysis (scanning and transmission electron microscopy) confirmed the modifications. Calibration curves in Cormay serum (CS) and selectivity tests with chronoamperometry were used to evaluate the biosensor’s performance. Non-linear ML regression algorithms for modelling glucose concentration and calibration parameters were tested to find the best-fit model for accurate predictions. The biosensor with BGr and enzyme encapsulation showed excellent performance with a linear range of 0.75–40 mM, a correlation of 0.988, and a detection limit of 0.078 mM. Of the algorithms tested, the decision tree accurately predicted calibration parameters and achieved a coefficient of determination above 0.9 for most metrics. Multilayer perceptron models effectively predicted glucose concentration with a coefficient of determination of 0.828, demonstrating the synergy of biosensor technology and ML for reliable glucose detection.