In this work, NiFe layered double hydroxides/β-Cyclodextrin/Chitosan (NiFe LDH/β-CD/CS) nanocomposite hydrogels are synthesized as innovative pH-responsive drug carriers via freeze and thawing method. Sunitinib malate (SUB) as a model of the anticancer drug was loaded in NiFe LDH nanosheets and different weight percentages of SUB@NiFe LDH (1 and 3 wt.%) were integrated into β-CD/CS hydrogel to enhance sustaining drug release behavior and the optimum value was obtained 3 wt.%. The structural characteristics of synthesized samples are examined using FTIR, XRD, SEM, TEM, and TGA analyses. In vitro SUB release from synthesized samples is greater at acidic pH equal to 5.5 than at neutral pH equal to 7.4. The cell viability of SUB@NiFe LDH/β-CD/CS nanocomposite hydrogel towards HT-29 cancer cells was obtained 45% after 72 h incubation showing a high cytotoxicity against these cancer cells. Also, drug-loaded nanocomposite hydrogels exhibited higher antibacterial activity towards S. aureus bacteria than E. Coli. Finally, the results suggest the developed nanocomposite hydrogels have a high potential to act as a biocompatible, pH-sensitive drug delivery system that can be leveraged for chemotherapy and other related therapeutic purpose.