The intrinsic birefringence of cellulose is one of the most fundamental optical parameters for analyzing and developing various cellulosic materials. However, the previously reported values greatly vary depending on the problems occurred due to the measured cellulose sample or method, and it is still a challenge to evaluate the intrinsic birefringence of cellulose using suitable cellulose samples and methodologies by taking account into the recent knowledge and techniques. Here, we estimated the intrinsic birefringence of cellulose to be 0.09 by a procedure with three valid factors: (1) bacterial cellulose nanofibers consisting of extended chain crystals of cellulose are used, (2) films with relatively small orientation degrees are fabricated, and (3) the in-plane retardation maps are measured. The resultant eigenvalue is greater than those reported for cellulose and many petroleum-based polymers. Therefore, cellulose could be used to develop high-performance light compensation films with large optical anisotropies for use in future optoelectronic devices.