We investigate the focus conditioning effects on molecular field-free alignment observed with high-order harmonic generation (HHG) from CO2 molecules. We also experimentally demonstrate that both the spectral shape and alignment signal of HHG significantly vary with changing focus position. A maximal alignment signal is achieved at a given focus position because of the optimal intensity of the driving laser. This intensity is related to the ionization potential of the molecules. These results indicate that a unique focus position provides an optimal alignment signal for practical applications.