A new biocompatible interface made up of hydroxyapitite-multiwalled carbon nanotubes (HA-MWCNTs) nanocomposite was developed for adhesion and electrochemical detection of human osteoblast-like cells (MG-63). The HA-MWCNTs nanocomposite was synthesized by self-assembling nano-hydroxyapitite onto multiwalled carbon nanotubes in solution, which was characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. The adhesion of the MG-63 on the nanocomposite film was studied by quartz crystal microbalance and cyclic voltammetry. The nanocomposites interface showed improved immobilization capacity for cells and good biocompatibility for preserving the activity of immobilized living cells. The living cells immobilized on an HA-MWCNTs modified glassy carbon electrode exhibited an irreversible anodic peak response being positively associated with the density and viability of cells, which was used to describe the cells growth and evaluate the effectiveness of antitumor drug 5-fluorouracil (5-FU) on the cells. The obtained 5-FU cytotoxicity results agreed well with those from conventional MTT assays. The highly biocompatible HA-MWCNTs nanocomposite is also expected to be an appropriate matrix for the electrochemical investigation of adhesion, proliferation, and apoptosis of many other relevant mammalian cells.