The first-line antituberculosis drug can cause hepatic injuries leading to acute liver failure and treatment discontinuation due to a lack of selective biomarker. Thus, the present study was designed to investigate whether Alu methylation was associated with anti-tuberculosis drug-induced liver injury (ATDILI) and could be an early ATDILI marker. Alu methylation levels in blood leucocyte of 47 tuberculosis patients with ATDILI, 48 tuberculosis patients with non-ATDILI, and 100 healthy controls were measured using quantitative combined bisulfite restriction analysis (qCOBRA). Methylation levels within Alu elements in the ATDILI cases were considerably reduced, compared with tuberculosis patients with non-ATDILI and healthy controls. In addition, hypomethylation of Alu was independently correlated with heightened susceptibility to ATDILI. Besides, multivariate linear regression analysis uncovered that Alu hypomethylation was independently associated with liver function indicators including aspartate transaminase and alanine transaminase evaluated within 60 days after initiation of the anti-tuberculosis drugs. Receiver-operating characteristic curve analysis revealed that Alu hypomethylation (AUC = 0.82) in peripheral blood leukocytes provided more specific and sensitive values than the original biomarkers such as aspartate transaminase and alanine transaminase, which cloud serve as a novel marker for differentiating tuberculosis patients with ATDILI from non-ATDILI within 7 days after treatment with the anti-tuberculosis drugs. Accordingly, Alu hypomethylation in blood leucocytes could be used as a novel biomarker for early ATDILI development.