ObjectiveTo explore the effects of Vitamin C treatment of constructing rabbit tibial periosteal cell sheets in vitro and characterize their basic biological characteristics. Methods The rabbit tibial periosteal cells were isolated and cultured in DMEM supplemented with 10% fetal bovine serum. The rabbit tibial periosteal cell sheet formation was observed under an inverted microscope. In order to assess the morphology of cells sheets and extracellular matrix, hematoxylin and eosin (H-E) and Masson staining were employed. The viability of the cell sheet was evaluated by the live and dead cell assay. Results Rabbit tibial periosteal cells could be successfully isolated and cultured in vitro. The result of CCK8 showed that the growth curve of cells was S-type. Crystal violet staining indicated that periosteal cells culturd in vitro had clony formation ability. And it possessed multifunctional differentiation potential. Furthermore, isolated cells could construct the rabbit tibia PCs sheet when incorporated with Vitamin C for 14 days. The constructed rabbit tibial periosteal cell sheets were translucent and composed of film-like structure. H-E and Masson staining showed that the cell sheets were predominant of rabbit tibial periosteal cells and abundant with ECM. When observed with live and dead fluorescence staining, cell sheets were presented with a higher percentage of live cells. Conclusion The rabbit tibia periosteal cell sheet can be successfully constructed by utilizing Vitamin C, which could be a novel path for bone defect repair.