The preparation of a highly oriented photoluminescent film of fluorescein (FLU) and 1‐heptanesulfonic acid sodium (HES) co‐intercalated in a layered double hydroxide (LDH) matrix by electrophoretic deposition (EPD) is reported, and its application as an optical pH sensor is demonstrated. The FLU‐HES/LDH films with thickness ranging from nanometer to micrometer on indium tin oxide substrates exhibite good c‐orientation of LDH platelets (the ab‐plane of the LDH platelets parallel to the substrate), as confirmed by X‐ray diffraction and scanning electron microscopy. Polarized luminescence of the film is observed with anisotropy value r = 0.29, resulting from the highly oriented FLU in the LDH gallery. Furthermore, the optical pH sensor with film thickness of 300 nm exhibits a broad linear dynamic range for solution pH (5.02–8.54), good repeatability (relative standard deviation (RSD) less than 1.5% in 20 consecutive cycles) and reversibility (RSD less than 1.5% in 20 cycles), high photostability and storage stability (ca. 95.2% of its initial fluorescence intensity remains after one month) as well as fast response time (2 s). Therefore, this work creates new opportunities for the preparation and application of LDH‐based chromophores in the field of optical sensors.