A cross-linking spacer molecule in biomaterials is an important factor in controlling their biocompatibility. The effects of various cross-linking spacers on the biocompatibility of chitosan-spacer-poly(ethylene oxide) (PEO) hydrogel were evaluated by various cytotoxicity assays and by targeting to specific cell organs. The chitosan-spacer-PEO hydrogel was synthesized using a Michael type addition reaction by employing chitosan-acrylate and PEO-thiols. Chitosan-acrylates have been synthesized employing three different cross-linking spacers such as 2-car-boxyethyl acrylate, linoleic acid and oleic acid. This study verified the grafting of the spacer molecules to the amine groups of chitosan side chains with FTIR by comparing the peaks of the chitosan-acrylates with those of the umnodified chitosan. After evaluating the cytotoxicity of the chitosan-acrylates in solution with a live a dead assay, the cytotoxicity of the chitosan-spacer-PEO gels was compared with each other by testing them with CCK-8 for their effects on cell proliferation, with a MTT assay for their effects on mitochondria damage, with BrdU assay for their effects on DNA damages and with the neutral red assay for their effects on lysosome damages. The degrees of cytotoxicity of the chitosan-spacer-PEO hydrogels were compared with those of Teflon and Latex, positive and negative control, respectively, using neural cells, such as PC-12 cells. The chitosan-2-carboxyethyl acrylate-PEO hydrogels demonstrated the best cell compatibility among the hydrogels employed in this study.