A novel diphenylalaninamid (FFA) based peptide nanoparticles (PNPs) modified pencil graphite electrodes (PGEs) for construction of electrochemical cytosensor was demonstrated for the first time in this study. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed the spherical nanostructure of the synthesized FFA based PNPs while attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectra provided information about the structure and conformation of proteins in their structure. Self assembly of PNPs on PGE surface and adhesion of DLD-1 cancer cells on this surface was also characterized by electrochemical measurements. PNP/PGE5 acted as a sensitive platform for simple and rapid quantification of low concentration of DLD-1 cancer cells in early diagnosis using the electrochemical impedance method (EIS). The offered cytosensor demonstrated outstanding performance for the detection of DLD-1 cells by the EIS method. The impedance of electronic transduction was associated with the amount of the immobilized cells ranging from 2 x 10(2) to 2.0 x 10(5) cells mL(-1) with a limit of detection of 100 cells mL(-1). The efficient performance of the cytosensor was attributed to the well-defined nanostructure and biocompability of PNPs on the substrate.