Neopterin is a catabolic product of guanosine triphosphate (GTP), a purine nucleotide. In human biological fluids such as urine, neopterin concentrations indicate the activation of cellular immune activation under the control of T helper cells. High neopterin concentrations in body fluids such as serum or urine also indicate the activation of cellular immunity, which is associated with the reaction to oxidative stress. In this work, neopterin is used as the target molecule and is then imprinted onto poly(ethylene-co-vinyl alcohol) via solvent evaporation. After removing the thin-film template molecules, the membrane is used as a sensing element for electrochemical urinalysis. Miniaturized devices were fabricated for continuous measurement of biomolecules in urine. A microfluidic channel made from polydimethylsiloxane (PDMS) is mounted on the electrochemical electrode chip. To maximize electrochemical response, the sensing area is a 30 × 1 mm2. In the random urine analysis, the cyclic voltammetry measurements of neopterin with additional recovery method achieved higher than 90% recovery gave the neopterin concentration 0.5 ng/mL.