A laser diffraction-induced dielectrophoresis (DEP) phenomenon for the patterning and manipulation of individual HepG2 cells and polystyrene beads via positive/negative DEP forces is reported in this paper. The optoelectronic substrate was fabricated using an organic photoconductive material, TiOPc, via a spin-coating process on an indium tin oxide glass surface. A piece of square aperture array grid grating was utilized to transform the collimating He-Ne laser beam into the multi-spot diffraction pattern which forms the virtual electrodes as the TiOPc-coating surface was illuminated by the multi-spot diffraction light pattern. HepG2 cells were trapped at the spot centers and polystyrene beads were trapped within the dim region of the illuminated image. The simulation results of light-induced electric field and a Fresnel diffraction image illustrated the distribution of trapped microparticles. The HepG2 morphology change, adhesion, and growth during a 5-day culture period demonstrated the cell viability through our manipulation. The power density inducing DEP phenomena, the characteristics of the thin TiOPc coating layer, the operating ac voltage/frequency, the sandwiched medium, the temperature rise due to the ac electric fields and the illuminating patterns are discussed in this paper. This concept of utilizing laser diffraction images to generate virtual electrodes on our TiOPc-based optoelectronic DEP chip extends the applications of optoelectronic dielectrophoretic manipulation.