Pullulan, a naturally occurring polysaccharide was oxidized using sodium periodate and converted into its aldehyde derivative and used as a bio crosslinker for stabilizing collagen. The crosslinking mainly occurred due to Schiff's base reaction between amino group of lysine or hydroxylysine present in collagen with the aldehyde groups of oxidized pullulan. A known concentration of collagen was crosslinked with different concentrations of oxidized pullulan and scaffolds were prepared by freeze drying method. The crosslinking efficiency and biodegradation of the crosslinked scaffolds showed higher degree of crosslinking and slower degradation rate with increase in cross linker concentration. The surface morphology of scaffolds using SEM showed that the scaffolds with 3% concentration of crosslinker (Col-OxP-3) exhibited highly porous nature with interconnecting pores of 66.35 μm size compared to other groups, which was also supported by porosity and tensile strength measurement. The tensile strength was 10 times higher in Col-OxP-3 scaffold compared to native collagen. The swelling capacity of the scaffolds increased with increase in crosslinker concentration and the equilibrium was retained up to 60 min. The crosslinked scaffolds were non-toxic to 3T3 fibroblast cell line proving their biocompatible nature. Thus, this new class of bio-crosslinker can be used in replacement of synthetic crosslinkers for biomaterial synthesis.