Abstract The boundaries between crystalline grains in hybrid perovskite films are beneficial for carrier dissociation, and may also serve as recombination centers to alter the photoluminescence property. Herein, it is shown that the grain boundaries can significantly enhance the anisotropy of photoluminescence from two‐dimensional (2D) hybrid perovskite films. The measurement of carrier densities shows that the grain boundaries generate an in‐gap state, which results in an additional red‐shifted photoluminescence band due to its emissive nature. Interestingly, the light emission of the in‐gap state appears only in the direction parallel to the film, corresponding to the in‐plane dipole transition as reveled by the measurement on photoluminescence polarization. These results demonstrate the tunable anisotropic photoluminescence from 2D perovskite materials toward the construction of directionally dependent light sources for advanced display application.