A facile microwave-assisted one-pot synthesis of sodium carboxymethylagarose and calcium carboxymethylagarose from Gracilaria dura agarose (Ag) has been described. The process is user friendly, and the highest degree of substitution was obtained within 15 min compared with the conventional method, which requires more than 3 h. Solubility and gelling behavior of the modified Ag products were found to be dependent on degree of substitution of the products. The characterizations were done by using Fourier transform infrared spectroscopy, 1H- and 13C-nuclear magnetic resonance spectroscopy, thermogravimetric analysis, scanning electron microscopy, Inductively coupled plasma spectrophotometry (ICP), rheology, conductometer analysis, and DNA gel electrophoresis. These agarose derivatives were easily soluble in water and exhibited low thermal hysteresis, improved conductivity, and improved the DNA resolution ability of the parent G. dura Ag hydrogels. These hydrogels may have potential applications in the areas including electrochemical devices, microbiology, biomedical, and pharmaceuticals fields. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40630.