Cryo-electron microscopy (cryo-EM) is a powerful technique capable of investigating samples in a hydrated state, compared to conventional high-vacuum electron microscopy that requires samples to be completely dry. During the drying process, numerous features and details may be lost due to damage caused by dehydration. Cryo-EM circumvents these problems by cryo-fixing the samples, thereby retaining the intact and original features of hydrated samples. This protocol describes a step-by-step cryo-scanning electron microscopy (cryo-SEM) experimental procedure with Chlorella sorokiniana as the subject. By employing filter paper as the sample substrate, we propose a simple and reliable method for cryo-fixation and freeze-fracture of Chlorella sorokiniana in water suspension. The advantage of using filter paper as a substrate lies in its ability to support a thin film of sample, enabling a cold knife to make a cut effortlessly and produce a clean freeze-fractured surface for SEM investigation. By following the approach described in this protocol, both the internal structure and surface morphology of Chlorella sorokiniana can be easily resolved with high quality. This protocol is highly versatile and can be applied to samples dispersed in water or solvents, including cyanobacterial cells, algal cells, and any kind of sample that can be adsorbed onto filter paper.