Background The simplex congenital blepharoptosis is the common blepharon motor dysfunction disease.Some researches have shown that congenital blepharoptosis is related to the hypoplasia of levator. Objective This study was to investigate the thickness and pathological features of levator palpebrae superioris aponeurosis in congenital blepharoptosis patients. Methods A prospective cohort study was carried out in Anyang Eye Hospital from March 2012 to April 2014.Eighty-five eyes of 56 patients with congenital blepharoptosis were divided into mild (15 eyes), moderate (25 eyes) and severe blepharoptosis (19 eyes) groups, and the fellow eyes of monocular blepharoptosis was used as fellow eye group (26 eyes). Twenty-six eyes of 13 normal subjects were recruited for the normal control group.The thickness of levator aponeurosis was measured by ultrasound biomicroscope (UBM), and the shifting range of levator aponeurosis was detected by using measuring scale.Levator aponeurosis specimens were collected during the levator palpebrae superioris shortening surgery for the pathological examination.The study was approved by the medical ethics committee of Anyang Eye Hospital, and the patients or their guardian signed the informed consent. Results The thickness of levator aponeurosis was (0.331±0.018), (0.373±0.026), (0.539±0.023), (0.557±0.024) and (0.547±0.028) mm in the severe blepharoptosis group, moderate blepharoptosis group, mild blepharoptosis group, normal control group and fellow eye group, respectively, showing a significant difference among them(F=1.681, P=0.043). The thickness values of levator aponeurosis were considerably lower in the severe blepharoptosis group and moderate blepharoptosis group than those in the mild blepharoptosis group, fellow eye group and normal control group (all at P<0.05), and the thickness value of levator aponeurosis was significantly reduced in the severe blepharoptosis group compared with the moderate blepharoptosis group (P<0.05). Pathological examination showed arranging disorder of muscle fibers, hyaline-like degeneration, connective tissue hyperplasia and interruption of endomysium.The number of eyes with severe hyaline-like degeneration and connective tissue hyperplasia was significantly increased in the severe blepharoptosis group than that in the moderate blepharoptosis group or the mild blepharoptosis group, as well as in the moderate blepharoptosis group than that in the mild blepharoptosis group(all at P<0.01). The adipose cells in muscle in the mild blepharoptosis group, moderate blepharoptosis group and severe blepharoptosis group were (12.35±4.62), (17.58±7.46) and (26.19±10.81)/field, and adipose cells in the severe blepharoptosis group were significantly more than those in the mild and moderate blepharoptosis groups (t=5.60, P=0.00; t=2.71, P=0.01). A significant increase in the adipose cells also was seen in the moderate blepharoptosis group compared with the mild blepharoptosis group (t=2.44, P=0.02). Conclusions UBM can offer accurate thickness data of levator aponeurosis.The combination of thickness data and shifting range measurement of levator aponeurosis is helpful for the evaluation of muscle strength.The development of levator aponeurosis appears to be abnormal in congenital blepharoptosis patients.The histopathological change parallels to the severity of the disease. Key words: Blepharoptosis, congenital; Biomeasurement; Ultrasound biomicroscopy; Levator aponeurosis; Histopathology