This work studied the structure and hybridization properties of DNA in simulated crowded media containing polyethylene glycol 10,000 and sucrose. Synchrotron radiation circular dichroism revealed that the overall CD profiles of a 21-mer and a d(CG)9 duplex remain similar in the simulated crowded fluids compared with those in Tris buffer, but the amplitude ratios of individual maxima and minima vary with the concentration of DNA and the media, suggesting possible conformational changes. Using fluorescence resonance energy transfer, this work confirmed that hairpin and duplex formations are not affected by the crowding condition. On the other hand, the duplex dynamics is affected by the crowding condition, where strand exchanges were promoted by the presence of PEG, but unaffected by sucrose.