We aim to investigate the potential of collagen extracted from rainbow trout for tissue engineering applications. In this regard, nanocomposite scaffolds based on the extracted collagen reinforced with various concentrations of boron nitride (BN) nanoparticles (0, 3, 6, 9, and 12 wt%) were developed. In addition, the role of various concentrations of BN nanoparticles and two-step cross-linking process on the physical and chemical properties of nanocomposite scaffolds were investigated. Our results demonstrated the isolation of Type I collagen with excellent thermal stability but with some structural and chemical differences compared to other sources. The synergic role of BN nanoparticles and two-step cross-linking process resulted in a noticeable improvement in the mechanical properties of collagen-BN scaffolds. Noticeably, incorporation of 6 wt% BN along with a two-step cross-linking process significantly increased the compressive strength (9.5 times) and elastic modulus (four times) of the collagen scaffold. Besides, nanocomposite scaffolds significantly improved proliferation and spreading of MG-63 cell line, confirming their biocompatibility. The results suggested that the incorporation of BN nanoparticles along with a two-step cross-linking process not only could promote the mechanical and thermal performances of collagen scaffolds, but also enhanced high cell viability, and proliferation supporting their potential in tissue engineering applications.