A novel breathable electrochemical enzyme-free sensor made from laser-induced carbon nanofibers embedding Ni nanocatalysts (Ni-LCNFs) is proposed for the capture and detection of biomarkers in breath aerosol. The permeable Ni-LCNF electrodes were fabricated on filter paper where a hydrophobic wax barrier was created to confine the device’s working area. The device was tested with aerosolized glucose, which was collected on the porous Ni-LCNF electrode. After a subsequent drying step, 0.1 M NaOH was dropped onto the device, and the electrocatalytic reaction of the captured glucose enabled by a Ni nanocatalyst was monitored via cyclic voltammetry (CV). Taking the oxidation/reduction peak ratios from CV as analytical signals improves the reliability and reproducibility of the glucose measurement. In the measurement step, closing the sensing area with adhesive tape, named closed device, enhances the detection sensitivity and enables the detection limit of 0.71 μM, which is 11.5 and 50 times, respectively, better when compared to the open device configuration. Measurements with simulated glucose aerosols containing clinically relevant glucose levels and comparison to screen-printed electrodes demonstrated the device’s superiority for breath analysis. Although in vivo validation studies must be conducted in future work, the proposed device results in a captivating point-of-care device integratable in breathing masks and breath analysis devices.