Current methods for detecting chronic airway inflammation, such as asthma, rely on complex procedures and specialized clinicians. Taking advantage of inherent nanomaterial properties and their chemical design flexibility, nanofibers were designed and integrated with enzyme entrapping hydrogels. This composition offers noninvasive sample collection followed by simple colorimetric detection. Specifically, nanofibers were made from positively charged nylon-poly(allylamine hydrochloride). They were optimized with respect to mat thickness, additive content, and lactate capture efficiency. The nanofibers could efficiently bind lactate through electrostatic interaction, correlating the resulting amount on the nanofiber mat to the concentration in breath aerosols. Detection was subsequently accomplished through a standard lactate oxidase, horseradish peroxidase assay with 3,3′,5,5′-tetramethylbenzidine colorimetric detection. The optimized nanofibers outperformed other polymeric nanofibers, face mask material, and filter paper regarding analyte capture and breathability due to the surface chemistry chosen and the high surface area afforded through the nanofiber mats. For lactate quantification directly on the mask, lactate oxidase was immobilized on the nanofiber mat via a hydrogel, ensuring long-term storage stability. Simple visual detection was achieved providing limits of detection of 5 μmol·L–1 (in solution) and 20 μmol·L–1 (hydrogel-based system) and a dynamic range that covers lactate concentrations found in breath, i.e., 5 to 150 μmol·L–1. This platform technology offers a promising solution for point-of-care diagnostics, contributing to remote healthcare, telemedicine, and simplified diagnostics in airway inflammation management.