In this study, combining the nanoparticle embedded sensors with lateral flow assays, a novel strategy for ensuring the quality of signalling in lateral flow assays (LFAs) was developed. A LFA for reactive oxygen species (ROS) is reported that is based on horse radish peroxidase (HRP) which is co-entrapped with Texas Red dextran inside porous polyacrylamide nanoparticles. In this system, enzymes are protected in the porous matrix of polyacrylamide which freely allows the diffusion of the analyte. The sensor is rapid and sensitive for quantification of hydrogen peroxide concentrations. A test solution of hydrogen peroxides was quantified with this novel LFA-ROS sensor to obtain a linear range between 1 and 25 μM. Nanoparticle embedding of enzymes is proposed here as a general strategy for developing enzyme-based lateral flow assays, eliminating adverse effects associated with biological samples.