The nanostructuration of biolayers has become a paradigm for exploiting nanoscopic light-matter phenomena for biosensing, among other biomedical purposes. In this work, we present a photopatterning method to create periodic structures of biomacromolecules based on a local and periodic mild denaturation of protein biolayers mediated by UV-laser irradiation. These nanostructures are constituted by a periodic modulation of the protein activity, so they are free of topographic and compositional changes along the pattern. Herein, we introduce the approach, explore the patterning parameters, characterize the resulting structures, and assess their overall homogeneity. This UV-based patterning principle has proven to be an easy, cost-effective, and fast way to fabricate large areas of homogeneous one-dimensional protein patterns (2 min, 15 × 1.2 mm, relative standard deviation ≃ 16%). This work also investigates the implementation of these protein patterns as transducers for diffractive biosensing. Using a model immunoassay, these patterns have demonstrated negligible signal contributions from non-specific bindings and comparable experimental limits of detection in buffer media and in human serum (53 and 36 ng·mL−1 of unlabeled IgG, respectively).