Gelatin derived from porcine sources raises significant ethical and religious concerns worldwide, particularly due to its increasing use in food-based products without adequate monitoring and control. Once gelatin is incorporated into processed food items, determining its source animal becomes challenging, which consequently increases the risks of adulteration and mislabeling. In this study, we propose the utilization of a novel nanocomposite comprising multi-walled carbon nanotubes (MWCNTs), chitosan (CS), and gold nano-urchins (AuNUs) for constructing a label-free electrochemical immunosensor. The purpose of this immunosensor is to effectively detect Porcine skin gelatin (PSG) in food samples using differential pulse voltammetry (DPV) with a 5 mM [K3Fe(CN6)] redox probe. Under optimized conditions, the fabricated biosensor demonstrates superior sensitivity to PSG, exhibiting a linear detection range of 50 pg/mL to 1000 a pg/mL and a limit of detection (LOD) of 23.079 pg/mL. Moreover, the constructed biosensor exhibits excellent reproducibility, acceptable selectivity, and interference resistance. Notably, it achieves outstanding recovery rates when applied to real food samples spiked with PSG, thus indicating its potential for practical implementation in food safety monitoring.