A novel electrochemical cytosensor was developed for detection of human osteosarcoma 143B cells based on graphene-three dimensional nanostructure gold nanocomposites (G-3D Au) using facile and rapid one-step electrochemical co-reduction technique. Characterizations by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that the gold nanoparticles in the scale of about 5-80 nm were deposited on the graphene sheets - showing pinecone-like 3D gold nanostructures. The proposed cytosensor, due to the increasing surface area and enhanced electron transfer rate by the nanocomposite, provided a highly stable and efficient matrix for the convenient conjugation of target CXCR4 antibody and the following incubation of cells. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) showed that the developed cytosensor was able to detect 143B cells ranging from 5.0 x 10(3) to 5 x 10(6) cells mL(-1) with a lower detection limit of 1292 cells mL(-1). This electrochemical cytosensor showed a fast response, high sensitivity and selectivity, which could be used in various applications such as cancer diagnosis, drug screening and cell adhesion studies.