Because of the toxic glutaraldehyde, the chitosan-glutaraldehyde hydrogels are seriously limited in biomedical applications. In this study, exploiting bioactive compounds of Aloe barbadensis Miller, the chitosan/ Aloe barbadensis Miller-glutaraldehyde (CS/AV-GDA) hydrogels were fabricated with 4-fold lower glutaraldehyde concentration without compromising hydrogel characteristics. The gelation time was controlled from a few seconds to several hours. The elastic modulus was varied from 483 to 99940 Pa. The CS/AV-GDA hydrogels could release the natural nitrite amount from 24.0 to 89.6 μM within the first hour for antibacterial activity, then continuously deliver a few μM every next hour for cell activities. The antibacterial test against Escherichia coli and Staphylococcus aureus revealed that the CS/AV-GDA hydrogels could kill the planktonic bacteria 5-fold more highly than control and prevent bacteria attachment on hydrogel surface effectively. Although the CS/AV-GDA hydrogels consumed only 0.25% glutaraldehyde concentration, their antibacterial capacities were comparable to chitosan-only hydrogels with 2% glutaraldehyde. For cytotoxicity tests, the CS/AV-GDA hydrogels using 0.25% glutaraldehyde concentration induce the human dermal fibroblasts proliferation significantly. All CS/AV-GDA hydrogels with glutaraldehyde crosslinker less than 1% showed non-cytotoxicity. As a result, the new CS/AV-GDA hydrogels might become an attractive candidate for medicine regeneration and tissue engineering.