A new fiber functionalization architecture for single‐fiber imaging and sensing is presented. 5(6)Carboxy‐seminaphthorhodafluor‐2 (a fluorescent pH sensor) is attached to a silk‐binding peptide and the complex added to aqueous silk fibroin protein. These bind with a Kd of 36 µM as determined by a fluorescence polarization assay. The fiber is dip‐coated into the silk and peptide mixture, and scanning electron microscopy images reveal a uniform silk coating on the fiber tip. The coating is stable to repeated washes and does not affect the imaging light emitted from the fiber, which allows concurrent optical coherence tomography (OCT) imaging and pH sensing. Oocytes are metabolically stimulated with CoCl2 to produce lactic acid, and a pH reduction of 0.04 is measured using the probe. The distance between fiber tip and oocyte is monitored by simultaneous OCT acquisitions to precisely position the probe. Lastly, OCT imaging of an ovary revealed the presence/absence of an oocyte within a follicle, an important step toward improving patient outcomes during in vitro fertilization, by limiting the number of invasive follicle punctures required. These results demonstrate the utility of this new coating to enable simultaneous OCT imaging and sensing, which provides significant insight into complex biological systems.

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