Biological materials are increasingly used in the repair of tissue defects. To reduce antigenicity, biological tissues have to be acellularized and or fixed with a crosslinking agent before implantation. In this study, an acellularization process was employed to remove the cellular components from swim bladder of fish (Labeo rohita) using 0.5% ionic biological detergent. The acellular matrix then was crosslinked with 0.6% glutaraldehyde (GA), 1% 1, 4-butanediol diglycidyl ether (BDDGE), and 1% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The physical properties of crosslinked samples were compared, in vitro, to those of the acellular counterparts on the basis of gross observations, degradation tests, free protein contents, free amino group contents, fixation index, free hydroxyproline contents determination, moisture percent and SDS-PAGE analysis. GA fixed sample showed highest resistance to nonenzymatic, collagenase degradation, and significant (P<0.05) decrease in free protein contents, free amino groups contents, and moisture percentage. SDS-PAGE analysis revealed that GA fixed acellular fish swim bladder expressed highest crosslinking, followed by BDDGE, and EDC treated tissues.