The biological properties of self cross-linked chondroitin sulfate (CS)-poly(ethylene oxide) (PEO) hydrogel including degradation, in vitro bone cell behaviors, and cellular compatibility were evaluated after the induction of its gel formation through use of different experimental conditions such as precursor composition, reaction temperatures, and precursor solution concentrations. The compositions of the CS-acrylate and PEO-thiol precursors had ratios of 25:75, 50:50, and 75:25. Hydrogel formations were induced with a mechanism of self cross-linking at the solution temperatures of 7, 23, 37, and 50℃ through use of different precursor solutions (2%, 3%, 5%, and 10%). Higher reaction temperatures and precursor concentrations induced quicker formation of the CS-PEO gel. While the 10% precursor solution became a hydrogel within 2 min at 50 ℃, the 2% solution took approximately 462 min at 7 ℃. While the CS-PEO gel with higher PEO content (25:75) swelled to 330%, the hydrogel with lower PEO content (75:25) swelled to 147% at 23 ℃, indicating that higher PEO content induced higher gel swelling. The CS-PEO hydrogel (50:50) gradually degraded in vitro through addition of 0.5 unit of chondroitinase ABC every 2 days, which led to approximately 7% degradation over 24 days. A CCK-8 assay performed on the hydrogel surface showed initially higher cell adhesion on the higher concentration CS gel (75:25), even though the adhered cells seemed to detach from their surfaces over time. Live and dead assays demonstrated that both cells on the surface of and inside the hydrogel were viable for 7 days. The results of in vitro bromodeoxyuridine, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and neutral red assays and evaluation of the gel covering on the cells cultured on the culture flask demonstrated excellent hydrogel cell compatibility (25:75, 50:50, and 75:25) compared to that of latex, the negative control.