In this paper, the feasibility of simultaneous electrochemical detection of glucose and non-esterified fatty acids (NEFA) is investigated using enzyme-based biosensors. The detection of glucose is based on ferrocene-Nafion-glucose oxidase (Fc-Nafion-GOx) modified screen-printed electrodes (SPEs). Solutions of Fc-Nafion-GOx were drop casted on SPEs and dried at room temperature allowing immobilisation of GOx entrapped in Nafion polymer with Fc. The detection of NEFA is based on bi-enzyme electrodes fabricated by the layer-by-layer technique applied onto SPEs using multilayers of poly(dimethyldiallyammonium chloride) polymer, multi-walled carbon nanotube composite films, and two enzymes: acyl-CoA synthetase and acyl-CoA oxidase. Oleoyl coenzyme A lithium salt (OACoA) and oleic acid (OA) were used as NEFA of interests because they are two of the most employed markers in clinical tests. Preliminary results demonstrated that enzyme electrodes showed linear response within the physiological concentration range of glucose and OA in human blood plasma as 5 and 1.2 mM, respectively. These results indicate a promising multi-biosensor platform for rapid one-step determination of glucose and NEFA simultaneously. This could lead the way toward the fabrication of glucose-NEFA biosensors, which can be important in diabetes management and early diagnosis.