The primary isolation and culture of penile corpus cavernosum cells are key techniques for studying the pathological mechanisms of erectile dysfunction (ED). This review summarizes the isolation and culture method of the major cell types in the penile corpus cavernosum, including smooth muscle cells, endothelial cells, pericytes, and fibroblasts. We discuss commonly used techniques, including enzymatic digestion, tissue explantation, immunomagnetic bead separation, and Matrigel induction, and compare their advantages and disadvantages. We also summarize the method for identifying different cell types. This systematic review of cell culture and identification techniques thus provides technical support and references for further research into the roles of penile corpus cavernosum cells in the onset, progression, and treatment of ED.