Milk is a highly nutritious food, and it is a source of necessary macro- and micronutrients for the growth, development and maintenance of human health. However, it may also be a source of food contaminants such as mycotoxins, pesticides and antibiotics that may cause disease. Amoxicillin (AMX) is one of the most frequently used lactam antibiotics in the world, the presence of its residues in milk poses a potential risk to public health. FDA and ECC 2377/90 have established the maximum residue limits (MRL) for AMX in milk to be 4 ng ml^-1. In recent years, nanofiber technology has opened up new horizons for the development of the biosensor to enhance the sensitivity, selectivity, and detection time. In this work, a novel ultrasensitive label-free electrochemical immunosensor for AMX has been developed. The immunosensor was fabricated by immobilization of anti-AMX on citric acid-grafted-Poly (vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membrane modified screen-printed electrode. PVA-co-PE nanofibrous membrane was prepared by electrospinning technique and characterized by scanning electron microscope (SEM) and the activation step was confirmed by Fourier transform infrared spectroscopy. The employment of PVA-co-PE nanofibers comparing with PVA-co-PE casted membrane and the successful fabrication steps were investigated by electrochemical impedance spectroscopy (EIS). The amperometric response measured at +0.65 V vs. the silver pseudo-reference electrode. Under the optimal conditions, the established immunosensor exhibited high sensitivity for AMX determination in a lower range of 0.009 – 10 ng mL^-1 with a determination limit of 7.5 pg mL^-1. The proposed immunosensor was evidenced to its applicability for AMX determination in milk samples without pretreatment, showing stability, reusability and good selectivity.