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