Glioblastoma is the most common malignant tumor in the central nervous system, with a generally poor prognosis mainly due to the limitations of surgical resection. Despite the introduction of fluorescence-guided surgical systems, which have increased the resection rate of glioblastoma to 65%, postoperative recurrence remains prevalent, partly due to the presence of highly invasive tumor cells, including tumor stem cells, at the infiltrated surgical margins. This review aims to explore the current application status, challenges, and potential improvements of 5-aminolevulinic acid (5-ALA) in enhancing the resection rate of glioblastoma surgery. We will analyze the mechanism of action of 5-ALA in fluorescence-guided surgery, discuss its ability to identify tumor cells at infiltrated margins, and explore how its effectiveness can be improved through fluorescence enhancement techniques during surgery. Furthermore, this article will also discuss the potential impact of 5-ALA on the prognosis and personalized treatment strategies for glioblastoma patients. By gaining a deeper understanding of the biological behavior and cellular characteristics of 5-ALA, we hope to provide more effective methods for surgical treatment of glioblastoma, thereby improving patient survival rates and quality of life.