The stabilization of metal nanoparticles is a key factor in various applications, but its wide use requires the development of effective and advanced materials. For the first time, this work demonstrates that in situ self-assembly of copper (Cu) and nickel (Ni) nanoparticles (NPs) via chitosan (Ct) allowed the formation of a new matrix Cu-Ct-Ni. The obtained Cu-Ct-Ni matrix was fully characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and UV–vis. The characterization results evidenced the ability of Ct chains to self-assemble Cu-NPs and Ni-Nps with a particular arrangement, forming a sunflower shape of 200–300 nm as diameter. Cu-NPs were stabilized outside Ct-chains, while Ni-NPs were located inside Ct, resulting in new nanomaterials Cu-Ct-Ni with higher thermal stability and unique morphology. The in situ self-assembly involved covalently cross-linked attraction and hydrogen bonding. Evaluation on the catalytic transformation of 4-nitrophenol to 4-aminophenol, Cu-Ct-Ni showed good stability and a high catalytic capacity. The assessed Cu-Ct-Ni nanocatalysts achieved an excellent conversion rate k of 0.719 cm−1 in 2 min with a turnover frequency (TOF) value of 11.55 s−1, making it the most effective and potential catalyst. The results help understand and analyze the catalytically active chitosan-bimetallic materials for environmental and medical applications.