Abstract The interaction of the classical intercalator ethidium bromide (EtBr) with the double helical network strands of DNA cryogels was investigated. The cryogels were prepared starting from aqueous solutions of DNA (about 2000 base pairs long) at −18 °C using 1,4-butanediol diglycidyl ether crosslinker under various reaction conditions. In contrast to the solubilization of DNA hydrogels in aqueous EtBr solutions, DNA cryogels remain stable even after complete saturation of their EtBr binding sites. The total binding capacity of the cryogels is 0.6 ± 0.1 EtBr per nucleotide, which is close to the theoretical maximum number of EtBr molecules that can bind to DNA. Even in very dilute solutions (down to 3 μM), cryogels remove EtBr from aqueous solutions with an efficiency of 90%. The equilibrium binding constant and the maximum number of EtBr binding sites of the cryogels almost coincide with the reported values for the secondary binding process of EtBr by DNA in aqueous solutions. At low mole ratios of bound EtBr to DNA, the cryogels swell with increasing amount of bound EtBr, most likely caused by the lengthening of DNA due to the intercalated EtBr. The response of DNA cryogels to changes in EtBr concentration between 3 and 300 μM also suggests that they can be used to detect DNA binding substrates in aqueous solutions.