This study presents a rapid and quantitative sequential separation method for H-3 and C-14 isotopes with distillation apparatus in environmental samples released from nuclear facilities. After adding 200 mg of granulated potassium permanganate and 500 mg of sodium hydroxide in 100 mL of sample solution, the sample solution was heated until approximately 10 mL of distillate, and the distillate fraction was removed. The sample solution was heated again until a minimum 10 mL of additional distillate was collected. 10 mL of distillate was transferred to the LSC vail and the measurement sample for H-3 was made by adding 10 mL of Ultima Gold LLT to the LSC vial. After adding 2.5 g of potassium persulfate, 2 mL of 1M silver nitrate and 15 mL of concentrated nitric acid to the remained sample solution, the sample solution was heated for 90 minutes and C-14 isotopes were adsorbed into 10 mL of Carbo-Sorb solution in glass vial. The measurement sample for C-14 was made by adding 10 mL of Permafluor to the C-14 fraction in glass vial. The purified H-3 and C-14 samples were measured by the liquid scintillation counter after quenching correction. The average recoveries of H-3 and C-14 with CRM were measured to be 96% and 85%, respectively. The sequential separation method for H-3 and C-14 investigated in this study was applied to activated charcoal filter produced from nuclear power plants after validating the reliability by result of proficiency test (KOLAS-KRISS, PT-2021-51).