Objective To investigate the retinal nerve fiber layer (RNFL) thickness of anisometropic amblyopic children with Littmann-adjusted OCT examination method. Methods A total of 30 anisometropic amblyopic subjects (4 to 14 years old) without treatment were enrolled, whose amlyopic eyes were amlyopic eye group and fellow eyes were fellow eye group. Also 50 emmetropic children′s right eyes were enrolled into normal group. Retinal nerve fiber layer thickness were obtained with optical coherence tomography (OCT), then were adjusted by Littmann formula. Each group was compared with other two groups specificly. Results Amblyopic eye group had no significant difference with other groups in each regions of RNFL ( P > 0 . 05 ) , both before and after Littmann-adjusted. The RNFL of amblyopic group group in T、 TI、 NI、 N regions had significant correlation with eye axis length before adjust(P < 0.05), while only T and NI had significant correlation after adjust (P<0.05). Conclusions The RNFL thickness of anisometropic amblyopic eyes had no significant difference with fellow eye and normal eye. Every research of RNFL thickness must consider the measurement error induced by eye axis length.