1,4-dioxane (DX) is a contaminant of emerging concern in water environments. The enrichment of DX-degrading bacteria indigenous to activated sludge is key for the efficient biological removal of DX in wastewater. To identify an effective substrate, which enables the selective enrichment of DX-degrading bacteria and has lower toxicity and persistence than DX, this study explored the effectiveness of tetrahydrofuran (THF) at enhancing the DX degradation ability of activated sludge without historical exposure to DX. Although the activated sludge initially exhibited negligible ability to degrade DX (100 mg-C/L) as the sole carbon source, the repeated batch cultivation on THF could enrich bacterial populations capable of degrading DX, inducing the DX degradation ability in activated sludge as effectively as DX did. The THF-enrichment culture after 4 weeks degraded 100 mg-C/L DX almost completely within 21 d. Sequencing analyses revealed that soluble di-iron monooxygenase group 5C, including THF/DX monooxygenase, would play a dominant role in the initial oxidation of DX in THF-enrichment culture, which completely differed from the enrichment culture cultivated on DX. The results indicate that THF can be applied as an effective substrate to enhance the DX degradation ability of microbial consortia, irrespective of the intrinsic ability.