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A flow-through biosensor has been developed and manufactured by 3D printing from poly(lactic acid). Uricase or tyrosinase were immobilized on the inner walls of the reactor cell and the concentrations of the reaction products, i.e., hydrogen peroxide in the case of uricase and quinone product of the tyrosine oxidation in the case of tyrosinase, were monitored in the amperometric mode. The screen-printed electrode modified with carbon black, pillar[5]arene and electropolymerized thionine and methylene blue was separated from the enzyme layer by 0.1 mm gap and used as a biosensor transducer. The components of the modifier layer exerted a synergetic effect and increased the cathodic current up to seven times compared to electrodes modified with individual components, increasing the sensitivity of the analysis. In the optimal conditions, biosensor allowed the determination of 1 × 10−7 to 1 × 10−5 M uric acid and 2 × 10−7 to 1 × 10−5 M tyrosine. The biosensors analyzed spiked samples of blood serum and urine and provided recoveries from 95 to 110%. Easy mounting of the flow-through cell and low cost of the replaceable parts make promising future application of the biosensor for routine clinical assays. [ABSTRACT FROM AUTHOR]