This study presents a synthesis method for environmentally friendly copper nanoparticles using ascorbic acid and gelatin as key components. The influence of precursor concentration, reductant amount, and stabilizer on the process was systematically investigated to obtain optimal results for the synthesis. The optimal parameters such as 20 g/L gelatin, 19.3 mM (AcO)₂Cu, and 41.5 mM ascorbic acid for forming copper nanoparticles were determined by central composite in the response surface design. Successful generation of pure copper nanoparticles with both spherical and cylindrical shapes, whose sizes were 43.1 and 105.2 nm, respectively, were confirmed by X-ray diffraction analysis and transmission electron microscopy. The synthesized nanomaterial was stable for a two-week storage time and then gradually oxidized into the form of Cu²⁺ ions. For antimicrobial activity testing, the synthesized nanoparticles displayed a distinctive ability to inhibit the growth of positive-gram bacteria (Lactobacillus fermentum, Bacillus subtilis, and Staphylococcus aureus), negative-gram bacteria (Escherichia coli), and cancer cells (A549, Hep-G2, KB, and MCF7). Copper nanoparticles synthesized chemical reduction had demonstrated notable inhibitory activity against various pathogenic fungi that affect plants, including Fusarium solani, Rhizoctonia solani, and Colletotrichum gloeosporioides. Additionally, the catalytic activity of the produced nanomaterial with a bandgap energy of 2.14 eV and a specific surface area of 40.6 m²/g was explored in the degradation of phenol, a common dye used in laboratories and industries. The optimization for phenol red removal reaching 94.4% after a 540-second reaction time was achieved using a response surface methodology, specifically a central composite design with an optimal dosage of copper nanoparticles at 31.5 ppm, NaBH₄ concentration of 53.1 mM, and pH = 7.5.