Novel hydrogel composed of both chondroitin sulfate (CS) and gelatin was developed for better cellular interaction through two step double crosslinking of -(3-diethylpropyl)--ethylcarbodiimide hydrochloride (EDC) chemistries and then click chemistry. EDC chemistry was proceeded during grafting of amino acid dihydrazide (ADH) to carboxylic groups in CS and gelatin network in separate reactions, thus obtaining CS-ADH and gelatin-ADH, respectively. CS-acrylate and gelatin-TCEP was obtained through a second EDC chemistry of the unreacted free amines of CS-ADH and gelatin-ADH with acrylic acid and tri(carboxyethyl)phosphine (TCEP), respectively. In situ CS-gelatin hydrogel was obtained via click chemistry by simple mixing of aqueous solutions of both CS-acrylate and gelatin-TCEP. ATR-FTIR spectroscopy showed formation of the new chemical bonds between CS and gelatin in CS-gelatin hydrogel network. SEM demonstrated microporous structure of the hydrogel. Within serial precursor concentrations of the CS-gelatin hydrogels studied, they showed trends of the reaction rates of gelation, where the higher concentration, the quicker the gelation occurred. studies, including assessment of cell viability (live and dead assay), cytotoxicity, biocompatibility via direct contacts of the hydrogels with cells, as well as measurement of inflammatory responses, showed their excellent biocompatibility. Eventually, the test results verified a promising potency for further application of CS-gelatin hydrogel in many biomedical fields, including drug delivery and tissue engineering by mimicking extracellular matrix components of tissues such as collagen and CS in cartilage.