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A novel method for spatially controlled layer-by-layer assembly of enzyme-based amperometric biosensors was developed for multi-analyte sensing with high spatial and temporal resolution. Stereo-electrodes with 75 μm tip separation and 1-3 μm tip size were functionalized using a simple three-step electrodeposition scheme to produce biosensors for simultaneous detection of glucose and lactate. Layers of nanostructured platinum, enzyme-doped conductive polymer poly(3,4-ethylenedioxythiophene) [PEDOT] and non-conductive polymer poly(o-aminophenol) [PoAP] were electrodeposited to achieve increased electroactive surface area, fine control over site and quantity of enzyme, and reduced interference. The resulting dual-electrode sensors showed good linear range, fast response times and negligible crosstalk. The proposed scheme can be easily expanded to multi-electrode systems and is adaptable to lab-on-chip platforms for a wide range of analytes.