Fused deposition modeling (FDM) represents a state‐of‐the‐art 3D printing technology that holds great promise in the field of electrochemistry as a fast, in‐house, and digital fabrication method of sustainable sensors. Despite the progress in FDM, existing practical 3D printed sensors still require post‐printing treatment to improve their operational features, either through (electro)chemical surface activation or surface modification with external functional materials. Herein, a novel lab‐made conductive biofilament with integrated two different types of metallic nanoparticles (NPs) for the 3D printing of ready‐to‐use and multiplexed electrochemical sensors is introduced. The filament is composed of biodegradable polylactic acid as a base, polyethylene glycol dimethyl ether as a plasticizer, carbon black as a conductive filler, and nanopowder oxides of bismuth and copper as integrated functional precursor materials. The as‐printed sensors serve as multifunctional transducers for the direct and sensitive determinations of several analytes, by exploiting the different properties of the two co‐existing NPs. Particularly, heavy metals (such as lead, and cadmium) are voltammetrically quantified by amalgamation with BiNPs electrogenerated from Bi2O3, while biomarkers (such as glucose, and uric acid) are determined in enzymatic‐free modes. Determination of glucose takes advantage of the electrocatalytic properties of CuO, while uric acid is directly oxidized at the sensor.