Dipstick-type lateral flow immunosensors are used widely for on-site detection of food allergens. The weakness of the immunosensors of this type, however, is their low sensitivity. Contrary to current methods, that focus on improving detection capability through the introduction of novel labels or multistep protocols, this work exploits macromolecular crowding to modify and regulate the microenvironment of the immunoassay, thus promoting the interactions that are responsible for allergen recognition and signal generation. The effect of 14 macromolecular crowding agents was explored using, as a model, commercially available and widely applied dipstick immunosensors, which are already optimized in terms of reagents and conditions for peanut allergen detection. An about 10-fold improvement in detection capability was achieved by using polyvinylpyrrolidone, Mr 29,000, as a macromolecular crowder without compromising simplicity and practicality. The proposed approach is complementary to other methods of improving the sensitivity by using novel labels. Because biomacromolecular interactions have a fundamental role in all types of biosensors, we foresee that the proposed strategy will also find applications in other biosensors and analytical devices.