Generally, chitosan-gelatin scaffolds were fabricated with 0.25 wt% glutaraldehyde (GA) as a cross linker by twice vacuum freeze drying. Three kinds of different mixture volume of chitosan and glutaraldehyde were considered. Apparent density, porosity, water absorption and compress properties of scaffolds with different freeze-dry temperature (-55°C or -75°C) were studied to obtain scaffolds with more excellent performances. It was found that porosity, water absorption and compress properties increased by adding volume of GA, however, apparent density decreased. With the dropping of freeze-dry temperature (from -55°C to -75°C), the porosity and water absorption of scaffolds decreased, but increased the apparent density and compress properties. It can be summarized that apparent density, porosity, water absorption and compress properties of scaffolds were influenced by changing the volume of GA in scaffolds and freeze-dry temperature. Thus, scaffolds with excellent performances would be expected to fabricate by adjusting volume of GA in scaffolds and freeze-dry temperature.