Growth of the vanadium pentoxide xerogel in the presence of the polyaniline thin film (V2O5/PANI) in different chemical treatment on substrate are presented. The in situ characterization studies revealed the presence of a lamellar structure for the V2O5/PANI hybrid material. The intercalation reaction was evidenced on the basis of the increase in the d-spacing as well as the displacement of the absorption bands toward lower energy levels. The growth of V2O5/PANI thin film, from direct reaction, on glasses substrate using pre-treated with cationic surfactant cetyl pyridinium chloride (CPC) and cetyl trimethylammonium bromide (CTAB) presented layers with a surface homogeneous. The UV/ozone and RCA treatment showed that the film had low adhesion on substrate compared with CPC and CTAB treatment. Furthermore, these results suggests that the CTAB and CPC treatment can be used, further, for V2O5/PANI LbL films using V2O5 gel as first layer as well as a promising candidate for applications as sensor for ammonia detection in poultry shed.