Copy number alterations (CNAs) are common in diffuse gliomas and have been shown to have diagnostic significance. While liquid biopsy for diffuse glioma has been widely investigated, techniques for detecting CNAs are currently limited to methods such as next-generation sequencing. Multiplex ligation-dependent probe amplification (MLPA) is an established method for copy number analysis in pre-specified loci. In this study, we investigated whether CNAs could be detected by MLPA using patients' cerebrospinal fluid (CSF). Twenty-five cases of adult diffuse glioma with CNAs were selected. Cell-free DNA (cfDNA) was extracted from the CSF, and DNA sizes and concentrations were recorded. Twelve samples, which had appropriate DNA sizes and concentrations, were subsequently used for analysis. MLPA could be successfully performed in all 12 cases, and the detected CNAs were concordant with those detected using tumor tissues. Cases with epidermal growth factor receptor (EGFR) amplification, combination of gain of chromosome 7 and loss of chromosome 10, platelet-derived growth factor receptor alpha amplification, cyclin-dependent kinase 4 amplification, and cyclin-dependent kinase inhibitor 2A (CDKN2A) homozygous deletion were clearly distinguished from those with normal copy numbers. Moreover, EGFR variant III was accurately detected based on CNA. Thus, our results demonstrate that copy number analysis can be successfully performed by MLPA of cfDNA extracted from the CSF of patients with diffuse glioma.