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We report reflective diffraction gratings made from smart hydrogels for ultrasensitive biochemical detection. As an example for a biochemically responsive hydrogel, we chose a pH-sensitive hydrogel to construct diffraction gratings that swell or shrink due to changes in pH. Interferometric analysis of the grating enabled detection of the hydrogel's swelling/shrinking with nanoscale precision with a resolution of 6 × 10-4 pH units. The developed system is remarkably simple both to fabricate and operate. Moreover, the concept of the hydrogel grating is generic and can be widely applied to hydrogels responsive to other stimuli.