In our previous conventional genome-wide association study (GWAS), WWOX was a susceptibility gene associated with acute lymphoblastic leukemia (ALL) development. Nowadays, advancements in genetic association analyses promote an in-depth exploration of ALL genomics. We conducted a two-step enrichment analysis at both gene and pathway levels based on ALL GWAS data including 269 cases and 1039 controls of Chinese descent. The following functional prediction and experiments were used to evaluate the genetic biology of candidate variants and genes. The serotonin-activated cation-selective channel complex gene-set was a potential biological pathway involved in ALL occurrence. Of which, individuals carrying the T allele of rs33940208 exhibited a prominent reduced risk of ALL [odds ratio (OR) = 0.71, 95% confidence interval (CI) = 0.53 to 0.96, P = 2.81 × 10], whereas those with the A allele of rs6779545 demonstrated an increased risk (OR = 1.23, 95% CI = 1.01 to 1.51, P = 4.11 × 10). Notably, the two variants displayed a better prediction capability when combined, that the risk of developing childhood ALL increased by 131% in subjects with 2-4 risk alleles compared to those with 0-1 risk alleles (P = 2.05 × 10). In addition, the T allele of rs33940208 could reduce HTR3A mRNA level, while the A allele of rs6779545 increased HTR3D mRNA expression. In this study, we identified HTR3A rs33940208 and HTR3D rs6779545 as potential susceptibility loci for ALL in Chinese children. Future validation and functional research will elucidate the underlying molecular processes, refining preventive strategies for this disease.