Pesticide and mycotoxin residues in food are concerning as they are harmful to human health. Traditional methods, such as high-performance liquid chromatography (HPLC) for such detection lack sensitivity and operation convenience. Efficient, accurate detection approaches are needed. With the recent development of nanotechnology, electrochemical biosensors based on nanomaterials have shown solid ability to detect trace pesticides and mycotoxins quickly and accurately. In this review, English articles about electrochemical biosensors in the past 11 years (2011-2022) were collected from PubMed database, and various nanomaterials are discussed, including noble metal nanomaterials, magnetic metal nanoparticles, metal-organic frameworks, carbon nanotubes, as well as graphene and its derivatives. Three main roles of such nanomaterials in the detection process are summarized, including biomolecule immobilization, signal generation, and signal amplification. The detection targets involve two types of pesticides (organophosphorus and carbamate) and six types of mycotoxins (aflatoxin, deoxynivalenol, zearalenone, fumonisin, ochratoxin A, and patulin). Although significant achievements have been made in the evolution of electrochemical nano-biosensors, many challenges remain to be overcome.