The primary cell isolation and culture of penile corpus cavernosum is one of the key technical methods for studying the pathological mechanisms of erectile dysfunction (ED). This review provides a detailed summary of the isolation and culture methods for major cell types in the penile corpus cavernosum, including smooth muscle cells (CSMCs), endothelial cells (CECs), pericytes (CPs), and fibroblasts (CFs). Commonly used techniques such as enzymatic digestion, tissue explantation, immunomagnetic bead separation, and Matrigel induction are discussed, with a comparative analysis of their advantages and disadvantages. Additionally, methods for the identification of different cell types are summarized. This systematic review of cell culture and identification techniques offers certain technical support and reference for further research into the roles of penile corpus cavernosum cells in the onset, progression, and treatment of ED.