The sensitive determination of folate receptors (FRs) in the early stages of cancer is of great significance for controlling the progression of cancerous cells. Many folic acid (FA)-based electrochemical biosensors have been utilized to detect FRs with promising performances, but most were complicated, non-reproducible, non-biocompatible, and time and cost consuming. Here, we developed an environmentally friendly and sensitive biosensor for FR detection. We proposed an electrochemical impedimetric biosensor formed by nanofibers (NFs) of bio-copolymers prepared by electrospinning. The biosensor combines the advantages of bio-friendly polymers, such as sodium alginate (SA) and polyethylene oxide (PEO) as an antifouling polymer, with FA as a biorecognition element. The NF nanocomposites were characterized using various techniques, including SEM, FTIR, zeta potential (ZP), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). We evaluated the performance of the NF biosensor using EIS and demonstrated FR detection in plasma with a limit of detection of 3 pM. Furthermore, the biosensor showed high selectivity, reliability, and good stability when stored for two months. This biosensor was constructed from ‘green credentials’ holding polymers that are highly needed in the new paradigm shift in the medical industry.