Human telomerase reverse transcriptase (TERT) is essential for the maintenance of telomere DNA length, chromosomal stability and cellular immortality. We hypothesized that TERT polymorphisms are associated with risk of childhood acute lymphoblastic leukemia (ALL). We first conducted a case-control study of 570 ALL cases and 673 cancer-free controls of Chinese children, using the tagging single-nucleotide polymorphisms (tSNPs) approach. We then examined the functionality of the important SNPs. We found that TERT promoter region tSNP (rs2735940) and two intron region tSNPs (rs2736100 and rs10069690) were associated with risk of childhood ALL (P = 0.036, 0.011 and 0.022, respectively, in allele comparison). The in vitro luciferase assays in Jurkat cells showed an increased transcriptional activity of rs2735940 T allele compared with the C allele. Additional experiments with ALL bone marrow revealed that the rs2735940 T allele increased levels of the TERT messenger RNA. Notably, TERT intron 2 polymorphism (rs2736100) was associated with lower telomerase activity and longer telomeres. Our findings suggested that TERT promoter rs2735940 polymorphism may affect the TERT activity, and rs2736100 may be associated with telomere function, and thus, it is a potential biomarker for genetic susceptibility to ALL in Chinese children.