<p>A simple and efficient method for producing lactate test strips was proposed, based on a one-step dropwise modification of screen-printed electrodes with three-component membrane-forming mixtures containing an enzyme, a mediator, and a polyelectrolyte. Compositions of mixtures for co-immobilization of lactate oxidase and mediators (potassium hexacyanoferrate(<span>iii</span>) or hexaammineruthenium(<span>iii</span>) chloride) in a chitosan membrane were developed. An increase in both the chitosan content (up to 0.1 vol.%) in the mixture and the signal registration time (up to 10–15 s) helped to expand the linear range of the test strips into the region of high lactate concentrations (up to 30 mmol L⁻¹). The applicability of the test strips for the lactate concentration determination in whole blood matrix was demonstrated, and the prospects for non-invasive lactate detection in sweat were considered. Thus, a method for manufacturing a biosensor was developed allowing simplification of the sensor production process and achieving optimal characteristics of biosensors for analyzing biological fluids.</p>