Objective To explore the morphological characteristics on cornea in patients with vernal keratoconjunctivitis(VKC)by the application of in vivo laser scanning confocal microscopy(LSCM).Methods The experimental design was retrospective observation case series(case control study).Twenty-six patients, each diagnosed as bilateral VKC, were enrolled in the study, among which 13 were tarsal form, 5 were bulbar form and the rest were mixed form. Nine patients had the clinical course less than one year, eight subjects longer than three years, and the rest between them. Another twenty-six healthy volunteers with matching age and gender were selected as normal control. All participants had their right eyes examined with the in vivo confocal microscopy ( HRT Ⅱ/RCM). Central cornea and superior peripheral cornea were chosen as the examination points. The images were recorded automatically and cellular density of each layer was analyzed by installed software. Software Image J was utilized to analyze the density, diameter, branch number and tortuosity of subbasal nerve fiber in VKC patients. Independent t test was performed to assess the differences on cellular density between VKC patients and normal control, as well as those between central and peripheral cornea in VKC patients. Fisher chi-square test was used to compare the infiltration rate of Langerhans cells in corneal epithelium between VKC patients and controls. ANOVA was applied to assess the differences in cellular density among three subtypes, as well as among different duration of VKC. Independent t-test and chi-square test were applied to analyze the parameters of subbasal nerve fiber. Results The morphological changes in cornea included the absence of superficial hyperreflective polygonal epithelial cells, infiltration of Langerhans cells in and(or) underneath corneal epithelium and activation of keratocytes in anterior stroma. Corneal epithelium conjunctivalization and stromal neovascularization could be identified in patients with corneal neovascular epithelium. Longitudinal or oblique dark striae could be found in the posterior stroma in patients with complicated keratoconus. The density of epithelial cells at central and peripheral cornea in healthy controls were (6033. 1 ± 998. 7) cells/mm2 and (6098. 4 ± 298. 3 ) cells/mm2, while that in VKC patients were (5972.2 ± 1148.2) cells/mm2 and (6178.5 ± 318.9) cells/mm2 respectively, the differences being no statistical significant between them (t = 1. 191 , 1. 011; P =0.238, 0. 318). However, it's found in VKC patients that cellular density at peripheral cornea was significantly higher than that at central area( t = 2. 249, P = 0. 03). The density of anterior stromal cells at central and peripheral cornea in healthy controls was (1001. 4 ± 125. 3) cells/mm2 and (924. 6 ± 201.4) cells/mm2, while that in VKC patients was (1184. 5 ± 115. 3 ) cells/mm2 and (1101.4 ± 151. 1) cells/mm2, the difference bearing no statistical significance(t =6. 617,3.439;P =0. 001). The density of posterior stromal cells in normal subjects and VKC patients was (537. 7 ± 42. 6) cells/mm2 and (548. 7 ± 79. 8) cells/mm2, that of endothelial cells was (2985. 7 ± 401. 2 ) cells/mm2 and (3021. 5 ± 383. 3) cells/mm2, respectively, neither difference had statistical significance (t = 0. 174, 1. 112; P = 0. 864, 0. 282 ) . Langerhans cell infiltration could be identified in 61.5% (16 cases) VKC patients, which was significantly higher than normal control (2 cases, 7. 7% ) (x2 = 12. 49, P = 0. 001 ). Furthermore, much intense Langerhans cells infiltration was found in bulbar form and mix form than tarsal form. (t = 6. 617, P = 0. 001). The density and diameter of subbasal nerve fiber in VKC patients decreased significantly than those in normal subjects, whereas the tortuosity increased significantly. Conclusions The morphological changes of cornea in VKC patients mainly involve corneal epithelium, subbasal nerve fiber and anterior stroma. In vivo LSCM is helpful in discriminating the subtypes of VKC.