Monolayer graphene has high symmetrical crystal structure and exhibits in-plane isotropic physical properties. However, twisted bilayer graphene (tBLG) is expected to differ physically, due to the broken symmetry introduced by the interlayer coupling between adjacent graphene layers. This symmetry breaking is usually accompanied by in-plane anisotropy in their electrical, optical and thermal properties. However, the existence of in-plane anisotropy in tBLG has remained evasive until now. Here, an unambiguous identification of the in-plane anisotropy in tBLG is established by angle-resolved polarized Raman spectroscopy. It was found that the double-resonant two-dimensional band is anisotropic. The degree of in-plane anisotropy is found to be dependent on the misorientation angles, which is two- and four-fold for tBLG with misorientation angles of 15° and 20°, respectively. This finding adds a new dimension to the properties of graphene, which opens a possibility to the development of graphene-based angle-resolved photonics and electronics.