Scaffold-guided tissue engineering based on synthetic and natural occurring polymers has gained much interest in recent years. In this article, the development of a polysaccharide-based artificial extracellular matrix (AECM) is reported. Three-dimensional, macroporous composite AECMs composed of chondroitin sulfate (ChS) and chitosan (Chito) were prepared by an interpolyelectrolyte complex/ lyophilization method. The ChS-Chito composite AECMs were crosslinked with glutaraldehyde and calcium ions (Ca2+) and cocrosslinked with N,N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The crosslinking reactions were examined with Fourier transform infrared analysis. Glutaraldehyde and Ca2+ crosslinked with Chito and ChS, respectively, to produce different types of ChS-Chito semi-interpenetrated networks. In contrast, EDC/NHS crosslinked with both Chito and ChS to produce ChS-Chito connected networks. In physiological buffer solutions, the Ca2+-crosslinked ChS-Chito composite AECMs showed a lower swelling ratio than their EDC/NHS- and glutaraldehyde-crosslinked counterparts. The ChS-Chito composite AECMs showed excellent antibacterial capability and biocompatibility according to the results of the in vitro antibacterial test and cytotoxic assay. This result suggested that the ChS-Chito composite AECMs might be a potential biomaterial for scaffold-guided tissue-engineering applications.