Introduction: The purpose of this study was to provide an optimum extraction method, purification, characterization and preliminary biological evaluation of type (I) fish scale collagen from fresh water Nile Tilapia (Oreochromisniloticas) fish, one of the most consumed fish species in Egyptian market, as a novel source of fish scale collagen, which can be effectively used in dental field. Materials and Methods: Nile Tilapia fish scales were washed, demineralized with HCl, extracted using pepsin / papain enzymes with two different concentrations for each, using two different methods. Characterization of the extracted collagen involved, examination of subunit composition using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Amino acid analysis using Amino-acid analyzer and gas chromatography mass spectrometry (GC/MS). Thermal analysis using Differential scanning calorimeter (DSC). Spectral analysis using Fourier transform infrared spectroscopy (FTIR). Determination of collagen fibril distribution and orientation using X-Ray diffraction analysis (XRD). In order to confirm the biocompatibility of produced gel, Baby Hamster kidney (BHK-21) fibroblast cells were seeded on 3D collagen gel. Results: Pepsin-solubilized collagen (PSC) was successfully prepared from the Nile Tilapia fish scales. The results of SDS-PAGE showed that extracted collagens were type I collagen. The denaturation temperature was (32 °C) .X-Ray Diffraction (XRD) analysis and Fourier-transformed infrared spectra (FTIR) showed that the extracted collagen had a triple helical structure. Active proliferation of BHK-21 cells with no signs of toxicity was evident on collagen gel. Conclusion: Nile Tilapia scales can be used as an effective source for type I collagen extraction. Nile Tilapia collagen fibrillar gel can be used at 37°C as a cellular matrix given by its melting temperature (42 °C). Collagen gel proved to be biocompatible, and is a promising potential biomaterial for biomedical applications.