Atomic force microscopy (AFM) has been widely applied in cellular morphology study. However, morphological information including volume and roughness obtained by AFM are usually affected by different kinds of factors, which include the microscopic system itself, imaging mode, or external factors such as AFM probe or tip condition. In this study, based on red blood cell model, the dependence of cellular morphology, volume, and roughness on several parameters of the imaging was evaluated and, furthermore, a general rule and resolution for trustful analysis had been suggested. In addition, the potential effects that resulted from sample itself had also been analyzed based on adhesive force analysis. The results indicated that the scanning range and the imaging mode affect cellular volume and roughness, and the distorted images should be ascribed to blunt tip, contaminated tip, and the shape of tip. The analysis of morphological distortion during AFM investigation of cells provides a reference for researchers using AFM.