A novel enzymatic method for extraction and preparation of fish collagen from swim bladder revealed the occurrence of α, β and γ bands with approximately 12.1 g/100g collagen corresponding to 89% of collagen and thus confirmed the nativity and purity of the fish collagen. FT-IR studies confirmed the retention of all three amide bands of I, II and III, and triple helixcity. UN-crosslinked and UV-crosslinked fish collagen membrane records a very high temperature of helix denaturation at 197˚C and 215˚C, shrinkage temperature at 50˚C ± 3.2˚C and 62˚C ± 2.7˚C and tensile strength at 16.89 ± 2.5 and 120.02 ± 1.0 Kg/cm 2 respectively. Fish collagen matrix promoted NIH 3T3 and L6 cellular growth and proliferation. The study indicates that availability of pure fish collagen could replace bovine collagen in tissue engineering applications.