BACKGROUND Central nervous system graft- vs -host disease (CNS-GVHD) is a rare cause of CNS disorders after allogeneic hematopoietic stem cell transplantation. Currently, establishing a diagnosis of CNS-GVHD is challenging because the diagnostic criteria and diagnostic methods are not well defined and many confounding factors need to be ruled out. CASE SUMMARY Here, we present two patients with CNS-GVHD. Both patients with a history of acute GVHD or chronic GVHD developed neurological symptoms that could not be explained by other causes, and had abnormal cerebrospinal fluid (CSF) studies as determined by CSF and blood immune biomarker examinations, suggestive of suspected CNS-GVHD. Due to the lack of specific magnetic resonance imaging abnormalities and the rapid clinical deterioration of the patients, we did not attempt to perform a brain biopsy, but prompted the initiation of empirical immunosuppressive therapy. In view of the rapid and favorable response to local and systematic immunosuppressive treatment and the aforementioned neurologic manifestations together with CSF abnormalities and other negative findings, a final diagnosis of CNS-GVHD was made. CONCLUSION CSF and blood immune biomarker examinations facilitated the diagnosis of CNS-GVHD, which are particularly suitable for patients who are critically ill and require urgent treatment and for those who are unsuitable for invasive diagnostic procedures.