Chemically cross-linked gelatin-chondroitin sulfate (ChS) hydrogels were prepared for the controlled release of small cationic proteins. The amount of chondroitin sulfate in the gelatin gels varied between 0 and 20 wt %. The chemical cross-link density, the degree of swelling, and the rheological behavior were determined to characterize the cross-linked hydrogels. Chemically cross-linked gelatin-ChS hydrogels were loaded with lysozyme, and the release was measured using phosphate-buffered saline. The lysozyme loading capacity of the hydrogels significantly increased with increasing chondroitin sulfate content of the gels. Compared to plain gelatin gels, the release rate of lysozyme slowed for the hydrogels containing 5 and 10 wt % of chondroitin sulfate, while the release was faster for hydrogels containing 20 wt % of chondroitin sulfate. The permeation of lysozyme through gelatin-ChS gels was measured using a two-compartment diffusion cell, and the effective diffusion coefficient was calculated. The effective diffusion of lysozyme in the gels was also qualitatively studied using fluorescence recovery after photobleaching. The Langmuir isotherms of lysozyme adsorption to gelatin-ChS gels and the lysozyme diffusion in the gels in the absence of electrostatic interactions were determined to evaluate the contributions of unspecific interaction between lysozyme and chondroitin sulfate and diffusion to the release. Both the interaction and the diffusion increase with increasing chondroitin sulfate content of the hydrogels, which resulted in a minimum value of the effective release rate for gels containing 5 wt % chondroitin sulfate.