Extracellular matrix (ECM) proteins, such as laminin and collagen, and their active peptides are potential candidates for affording cell adhesiveness to non-cell adhesive materials for three-dimensional (3D) cell culture. We have identified various biologically active peptides from laminins using more than 3,000 peptides. These peptides specifically interact with various cell surface receptors including integrins, syndecan, and α-dystroglycan. We have demonstrated that the cell adhesive peptides conjugated polysaccharides (chitosan, alginate, and hyaluronic acid), peptide-polysaccharide matrices, can mimick the biological function of ECM and are useful as a functional biomaterial. The cell adhesive peptides have the potential ability to serve as bio-adhesiveness for 3D cell culture matrices. Previously, we demonstrated that “mixing” of syndecan-binding peptides, such as AG73 (RKRLQVQLSIRT), provide cell adhesiveness to agarose gels while integrin-binding peptides, such as A99 (AGTFALRGDNPQG), do not. In this study, to conjugate the laminin peptides to agarose, we synthesized agarose-aldehyde by oxidation of primary alcohols of agarose using TEMPO reagent. Two laminin peptides, AG73 and A99, were synthesized with a CGG sequence at N-terminus and conjugated to agarose-aldehyde via thiazolidine formation between the cysteine residue and the aldehyde. Two-dimensional cell adhesion assay using fibroblasts showed that AG73- and A99-conjugated agarose gels have potent cell adhesion activity. Cell encapsulation experiments revealed that only the A99-agarose gels promote elongation and proliferation of fibroblasts in a 3D environment. These results suggest that covalent conjugation of the peptides to agarose is more beneficial to develop a peptide/agarose matrix compared to the previous mixing method, and the peptide-conjugated agarose gels are promising as a matrix for 3D cell culture for anchorage-dependent cells.