Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive B-cell lymphoma in which early diagnosis remains challenging. Although cerebrospinal fluid (CSF) B-cell-associated factors including soluble interleukin-2 receptor subunit alpha (IL-2RA) are known diagnostic markers, they often reflect neuroinflammation and are insufficient on their own to reliably differentiate PCNSL from neuroimmunological diseases. On the other hand, CSF immune checkpoint molecules reflect neuro-immune regulation and remain incompletely evaluated as biomarkers for PCNSL. We aimed to determine whether CSF immune checkpoint molecules can serve as additional diagnostic and prognostic biomarkers for PCNSL alongside B-cell-associated factors. Methods: In this retrospective cohort study, CSF samples from 33 patients with PCNSL and 85 patients with control diseases, including neuroimmunological diseases and other brain tumors, were analyzed using a bead-based multiplex immune-assay. Correlations between markers were evaluated using Spearman’s rank correlation and hierarchical clustering. Diagnostic performance was assessed using logistic regression modeling. Progression-free survival (PFS) was analyzed using the Kaplan–Meier method. Results: CSF levels of B-cell-associated factors (interleukin-10, C–X–C motif chemokine ligand 13, and IL-2RA) and immune checkpoint molecules (Fas ligand, T-cell immunoglobulin and mucin domain 3 (TIM-3), and galectin-9) were significantly elevated in PCNSL compared with control diseases. Correlation and cluster analysis identified two distinct marker clusters: B-cell-associated factors and immune checkpoint molecules. Two-marker diagnostic models combining IL-10 and immune checkpoint molecules demonstrated superior diagnostic performance. Higher levels of B-cell-associated factors showed a non-significant trend towards shorter PFS. Conclusions: CSF TIM-3 and galectin-9 represented additional diagnostic biomarkers for PCNSL alongside B-cell-associated factors.